MICROSCOPY RESEARCH AND TECHNIQUE 22:225-264 (1992)

Cortical Ultrastructure and Chemoreception in C iated Protists (CiI iophora) LINDA A. HUFNAGEL Department of Microbiology, University of Rhode Island, Kingston, Rhode Island 02881

KEY WORDS

Protozoa, Electron microscopy, Cilia, Membrane, Chemosei Dry, Olfaction, Chemotaxis

ABSTRACT

The ciliated protists (ciliates) offer a unique opportunity to explore the relationship between chemoreception and cell structure. Ciliates resemble chemosensory neurons in their responses to stimuli and presence of cilia. Ciliates have highly patterned surfaces that should permit precise localization of chemoreceptors in relation to effector organelles. Furthermore, ciliates are easy to grow and to manipulate genetically; they can also be readily studied biochemically and by electrophysiological techniques. This review contains a comparative description of the ultrastructural features of the ciliate cell surface relevant to chemoreception, examines the structural features of putative chemoreceptive cilia, and provides a summary of the electron microscopic information available so far bearing on chemoreceptive aspects of swimming, feeding, excretion, endocytosis, and sexual responses of ciliates. The electron microscopic identification and localization of specific chemoreceptive macromolecules and organelles a t the molecular level have not yet been achieved in ciliates. These await the development of specific probes for chemoreceptor and transduction macromolecules. Nevertheless, the electron microscope has provided a wealth of information about the surface features of ciliates where chemoreception is believed to take place. Such morphological information will prove essential to a complete understanding of reception and transduction at the molecular level. In the ciliates, major questions to be answered relate to the apportionment of chemoreceptive functions between the cilia and cell soma, the global distribution of receptors in relation to the anterior-posterior, dorsal-ventral, and left-right axes of the cell, and the relationship of receptors to ultrastructural components of the cell coat, cell membrane, and cytoskeleton. o 1992 Wiley-Liss, Inc.

INTRODUCTION Ciliates and the Problem of Chemoreception by Cells Electron microscopy can provide information about the functional anatomy of chemoreceptive systems at many levels of organization. Yet even for the most intensively studied invertebrate and vertebrate systems, what is known a t the subcellular level about the ultrastructural correlates of olfaction remains limited. If we broadly define chemoreception to include perireceptor events, ligand binding, transduction processes, and transmembrane ionic movements via ion pumps and channels, then ultrastructurally there is much to be revealed about the subcellular locale of each of these activities, for numerous different chemical signals. Why has so little information been forthcoming? Most likely it is due in part to the technical problems facing those who work with the multicellular chemoreceptive systems of higher organisms. Another reason is that very few chemoreceptor molecules appear to be needed for most responses, due to intervening signal amplification processes. The molecules that mediate chemoreception therefore have been very difficult to find by conventional biochemical and ultrastructural methods. Finally, only recently have the methods and specific probes been developed that will provide the specificity

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and resolution required to convincingly localize and ultrastructurally characterize the molecular machinery of chemoreception. The ciliated protists [“ciliates”; “cilioprotists” (Heywood and Rothschild, 198711, members of the phylum Ciliophora (see Fig. 1; Table 1) in the kingdom Protista, offer a unique opportunity to take advantage of emerging technological advances, to explore the relationship between chemoreception and cell structure. Ciliated protists are unicellular eukaryotes generally having numerous motile cilia, two types of nuclei (somatic and gametic), and a conjugation-type sexual reproductive habit. Because they are easy to grow in quantities sufficient for biochemical and molecular biological studies, readily analyzed genetically, and large enough for electrophysiological measurements, a variety of ciliates have been popular experimental organisms for studies of cellular physiology and behavior. Ciliates have been likened to neurons because they respond to various stimuli by exhibiting graded action

Received April 4, 1991; accepted in revised form July 30, 1991. Address reprint requests to Linda Hufnagel, Ph.D., Department of Microbiology, Morrill Life Science Building, University of Rhcde Island, Kingston, RI 02881.

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GYMNOSTOMATA Fig. 1. One author’sview of the phylogenetic relationships which may exist among various groups of ciliates. The purpose of this figure is to give an impression of the range of diverse morphotypes found among the ciliates. Since the systematics of the ciliates are rapidly evolving, some of the relationships illustrated may no longer be considered correct by some systematists. Upper case: subclasses (toconform to Table 1, change the “a” suffx to “ia”; lower case: orders. (Reproduced from Bardele, 1981, with permission of Elsevier Scientific Publishers Ireland Ltd.)

potentials which mediate behavioral responses (cf. Hinrichsen et al., 1985). For this reason, ciliates have been recognized as especially suitable, perhaps ethically superior in some instances (Hennessey, 1989b), for investigating properties of the ion channels that mediate behavior of cells. Ciliates are small in size, (10-200 pm), which makes them particularly amenable to detailed ultrastructural analysis. Some ciliate species, such as Paramecium tetraurelia, Tetrahymena thermophila, Euplotes raikovi, Stylonychia lemnae, and Oxytricha nova, have become the targets of intense cellular and molecular genetic analysis (cf. Gall, 1986). Thus, ciliated protista are especially attractive subjects for analysis of basic cellular principles underlying chemoreception. A major problem of chemoreception is to identify the structures and localize the macromolecules which serve as receptors and transducers for chemical stimuli acting at the cell surface. As we shall see, ciliated pro-

tista have highly developed surface patterns that can help to pinpoint chemoreceptive structures and macromolecules in relation to the effector organelles responsible for cellular responses. [For an up-to-date ultrastructural review of the Ciliophora, see Lynn and Corliss (1991).I The convincing ultrastructural demonstration of chemoreceptors in ciliates requires that the chemoreceptor proteins be clearly identified and characterized. It is interesting to note that this has been most successfully accomplished in unicellular organisms (Van Haastert et al., 1991; Van Houten et al., 1991). The greatest progress has been with Dictyostelium and yeast, but the cyclic AMP receptor molecule of Paramecium is now also on the verge of being characterized (Van Houten et al., 1991), making it likely that this receptor will soon be localized in Paramecium by electron microscopy, and promising to thrust ciliates into the forefront of chemoreceptor study.

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TABLE 1. Systematic organization of the phylum Ciliophora (ciliated protists) based on the Leuine Report (1980)of the Society of Protozoologists (Lee et al., 19851,showing placement of the genera (italicized) discussed in this reuiew, and subclasses and orders illustrated in Figure 1 (*I'

TABLE 1. Systematic organization of the phylum Ciliophora (ciliated protists) based on the Levine Report (1980)of the Society of Protozoologists (Lee et al., 19851,showing placement of the genera (italicized) discussed in this reuiew, a n d subclasses a n d orders illustrated in Figure 1 (*) (continued)'

Phylum Ciliophora Subphylum Postciliodesmatophora Class Karyorelictea Order Protocruziida Order Protostomatida Tracheloraphis Order Loxodida Order Protoheterotrichida* Class Spirotrichea Subclass Heterotrichia* (Spirotricha) Order Licnophorida Order Armophorida Order Odontostomatida* Order Clevelandellida Order Heterotrichida* Stentor Spirostomum Blepharisma Eufolliculina Order Plagiomida Order Phacodiniida Subclass Choreotrichia Order Choreotrichida* (Tininnida) Tintinnopsis Order Oligotrichida* Strombidium Subclass Stichotrichia Order Stichotrichida* (Hypotrichida) Oxytricha Stylonychia Subphylum Rhabdophora Class Prostomatea Order Prostomatida* Order Prorodontida Placus Class Litostomatea Subclass Haptoria Order Pleurostomatida* Loxophyllum Litonotus Order Pharyngophorida Dileptus Order Haptorida Didinium Lagynophrya Spathidium Homalozoon Bryophylum Enchelydium Subclass Trichostomatia* (Trichostomata) Order Vestibuliferida Balantidium Order Entodiniomorphida* (Entodiniomorpha) Ophryoscolescidae (family which includes the genera Entodinium, Ophryoscolex, and Eudiplodinium) Subphylum Cyrtophora Class Phyllopharyngea Subclass Phyllopharyngia Order Cyrtophorida* Order Rhynchodida* Subclass Chonotrichia* (Chonotrichida) Order Cryptogemmida Order Exogemmida Spirochona Subclass Suctoria* Order Exogenida Order Endogenida Tokophrya Order Evaginogenida Discophrya

Phylum Ciliophora (continued) Subphylum Cyrtophora (continued) Class Nassophorea Subclass Nassophoria Order Synhymeniida Order Nassulida* Order Microthoracida Order Propeniculida Pseudomicrothorax Order Peniculida Paramecium Frontonia Subclass Hypotrichia Order Euplotida Euplotes Certesia Aspidisca Class Oligohymenophorea Subclass Hymenostomatia* (Hymenostomata) Order Hymenostomatida* Tetrahymena Glaucoma Order Scuticociliatida* Cyclidium Subclass Peritrichia* (Peritricha) Order Sessilida Vorticella Zoothamnium Epistylis Order Mobilida Subclass Astomatia* (Astomata) Subclass Apostomatia Order Pilisuctorida Order Astomatophorida Order Apostomatida* Hyalophysa Terebrospira Subclass Plagiopylia Order Plagiopylida Class Colpodea Order Colpodida* Bresslaua Colpoda Tillina Order Bursariomorphida Order Bryophryida Order Cyrtolophosidida Order Sorogenida Sorogena

(continued)

'Variants of names used in Figure 1 are shown in parentheses

Ciliated protists provide a special opportunity to explore the role of cilia and flagella in chemoreception. A prominent feature of chemosensory neurons in both vertebrates and invertebrates is the presence of a specialized sensory apparatus having either cilia/f lagella, microvilli (sometimes called stereocilia), or both. The cilia of ciliates are homologous with the cilia and flagella of multicellular organisms, including those found on neuronal sensory cells. However, ciliate cilia are easier to study because they can be isolated abundantly and in extremely pure form from cells grown in axenic mass cultures. In olfactory neurons, it is commonly held that the physiological processes normally accompanying ciliary/flagellar motility are somehow harnessed to permit the reception and transduction of

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chemical stimuli (cf. Moran and Rowley, 1983). In vertebrate taste buds, on the other hand, the chemoreceptor cells do not have cilia or flagella; their appendages are specialized microvilli. Ciliates do not usually have stereocilia or other microvilli-like appendages. The cilia themselves or the surrounding somatic cell membranes must be the sites of chemoreception and transduction. In multicellular organisms, the chemoreceptive properties of cilia and flagella present a confusing picture. Receptors for some chemical stimuli reside on cilia, e.g., those for sex hormones in the wild silkmoth Antheraea polyphemus (Kaissling, 1986; Vogt et al., 1988) and odorant receptors in olfactory epithelial of fish and mammals (Rhein and Cagan, 1980, 1981). Nevertheless, there are other instances where the subcellular locale of chemoreception is not on cilia or is unknown. As we shall see, the situation with the unicellular organisms is just as complex. In a few cases, e.g., mating interactions of the flagellated protist Chlamydomonas (reviewed by van den Ende et al., 1990), it is clear that protistan cilia play a central role in reception and response t o chemical stimuli; in other cases, e.g., cell interactions during mating in the cilioprotist Tetrahymena (Hufnagel, unpublished), the evidence is compelling but not conclusive. Then there are those instances where cilia probably have few if any chemoreceptors, e.g., for cyclic AMP in Paramecium (cf. Preston and Van Houten, 1987b; see below). However, the ease with which extremely pure ciliary fractions can be obtained from ciliates such as Paramecium and Tetrahymena, and the fact that some ciliates are extremely amenable to molecular and genetic analysis, make it very likely that ciliated protists will be among the first cells in which the electron microscope will help to demonstrate convincingly the precise location of functional chemoreceptor molecules, particularly in relation to ciliary and cell soma membrane domains. Since there is already good evidence that in ciliates all parts of the cell surface have chemoreceptive activity, this review examines the ultrastructural properties of all cell surfaces, both ciliary and somatic, which may have chemoreceptive activity. While definitive ultrastructural studies localizing chemoreceptors, transduction molecules, and receptorrelated ion channels have not yet been reported for ciliates, the field is moving rapidly and such studies may appear very soon. In the meantime, electron microscopy can provide an anatomical perspective on chemoreception. Anatomical evidence for chemoreceptor function, based on analogy and deductive reasoning, can help to focus attention on certain structures and t o formulate the questions that need to be asked. A detailed structural description of the cortex of the ciliate cell can show where the organelles which respond to chemical stimuli reside and suggest who the players might be in chemoreception, chemotransduction, and chemoresponse. We presume that the ability to respond to environmental chemical cues preceded the evolution of the protistan cell, since modern bacterial cells have chemical senses (Adler, 1975). Nevertheless, ciliates are believed to have had a long evolutionary history, possibly

having diverged from other groups more than 400 million years ago (Lynn and Corliss, 1991; Nanney, 1985). It is possible, therefore, that ciliate chemoreception will have some unique aspects, reflected in ultrastructure and recorded in the genome. To fully understand the message in the genes, it may also be necessary to read the ultrastructure. In addition to chemicals, ciliates respond to touch, gravity, and light. Some of the molecular equipment used for chemoreception may be utilized for other types of reception as well, especially at the level of stimulus transduction. So, in our search for the ultrastructural correlates of chemoreception, we will consider the extensive information available on mechanoreception in ciliates.

Responses to Chemical Stimuli in Ciliates It is helpful for discussion purposes to classify ciliate chemoreceptive activity according to the physiological responses which are elicited, even though these systems are likely to overlap. Six broad categories can be defined: 1)motility responses leading to accumulation or avoidance; 2) feeding responses leading to selective nutrient uptake; 3) sexual responses leading to cell pairing and gene exchange; 4) exocytotic secretory responses leading to extrusome discharge; 5) settling responses leading to substrate selection and attachment; and 6 ) host invasion responses. Later this review focuses sequentially on several of these response categories. Molecular Substances Eliciting Responses in Ciliates Van Houten et al. (1981) assembled an impressive list of chemical cues to which ciliates respond, including signals used in mate recognition, host invasion, and food recognition. Diverse and numerous kinds of substances are represented, from inorganic ions to fish slime. As discussed recently by Machemer (19891, chemoresponses in ciliates appear to be mediated by modulations of membrane potential and conductances which can come about via two distinct processes: 1) generalized effects of changes in inorganic electrochemical gradients, and 2) specific signaling by ions and molecules, presumably through binding to cellular receptors. For this reason, the role of inorganic ions in chemoreception is likely to be particularly complex. Therefore, this review focuses on those chemoreceptive processes where the molecular signals are believed to be organic molecules and appear to act externally by binding to the cell surface. Such functional, ligandbinding molecules will be called receptors and the molecular assemblies of which they are a part will be referred to as receptor modules. The latter are taken to include the receptor molecules themselves, as well as associated macromolecules such as the G proteins that function in signal transduction, and ion channels, such as calcium channels. These molecules and modules are expected to reside in the plasma membrane and the external glycocalyx (cell coat), so this review focuses on these two cell components.

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

Anatomical Constraints of Chemoreception in Ciliates What factors might influence the distribution of chemoreceptors on the ciliate cell surface? The need t o communicate efficiently with effector organelles may impose anatomical restrictions on receptor distribution. For example, one would expect some chemoreceptors to be located close to or on cilia and other motility organelles and others to be localized in the vicinity of organelles used for food capture, substrate selection, and other behaviors. Other receptors may be localized to cell surfaces which are most likely to come into contact with a chemical stimulus, e.g., the anterior end of the cell or the tips of the cilia. However, some chemicals probably reach all parts of the cell surface at about the same time and proximity may not always be necessary for effective communication between receptors and effector organelles. Thus, some receptors may show a more globally uniform distribution. This may be true of receptors mediating chemokinetic motility responses ofParamecium and Tetrahymena (Almagor et al., 1981; Dryl, 1973; Levandowsky and Hauser, 1978; Van Houten, 19781, since these receptors may detect changes in concentration of the chemical moving past the cell by sampling a t different times. On the other hand, we cannot rule out the possibility that such receptors are unevenly distributed, until suitable ultrastructural studies are done. Where electron microscopy reveals an unexpected distribution of receptors, this may generate new ideas about how receptor function is integrated into overall cellular behavior.

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changes in tertiary or quaternary conformation of chemoreceptor molecules correlated with functional changes, and to identify reactive sites and antigenic sites on receptor molecules. The newest technologies, such as scanning-tunneling microscopy (cf. Amrein et al., 1989) and spot-scan imaging for transmission electron microscopy (TEM) (Downing, 1991) may soon permit the analysis of receptor-rich membrane regions under physiological conditions. As we shall see, the literature is not yet overburdened by reports of experiments focused directly on ultrastructural parameters of chemoreception in ciliates. But this is the basis for hope that future efforts in this direction will lead to the discovery of new paradigms for how cells sense and respond to chemicals in their environment.

CILIATE ULTRASTRUCTURE General Features The ciliated protist is both a cell and a complete organism. Within a single cell are contained all the components required for reception of and response to chemical stimuli. Like other cells, the ciliate consists of a complex and dynamic three-dimensional cytoskeletal scaffold supporting various membranous vesicles, cisternae, and self-reproducing organelles, separated from the environment by a continuous and often dynamic multilayered sheet, composed of the plasma membrane and cell coat. At best, the electron microscopist presently can expect to view only snapshots of the structure and activity of this complex system. Fortunately, a t the cell surface where chemosensory activity is expected to occur, the ciliate cytoskeleton is extremely stable in comparison to many other cells. Furthermore, ciliates exhibit a highly repetitive, defined cortical structure, based on the ciliary unit (see below). This organization and stability, together with well-defined morphogenetic processes which accompany various developmental events such as cell division, provide a framework for defining the anatomical parameters of chemoreception.

How Can the Electron Microscope Contribute? In addition to providing a general anatomical description of putative chemoreceptive organelles, the electron microscope can corroborate the existence of receptor molecules a t the cell’s surface, show where they reside, and determine whether they are fixed or mobile. Ultrastructural approaches can also reveal how receptors are organized to make signal capture most efficient, trace the fate of receptors that have inCiliary Unit teracted with the chemical stimulus (whether they remain at the cell surface, slough off the cell, or are inThe surface structures of ciliates comprise the “pelternalized), identify structures that serve to interpret licle” or “cortex” and include (from outside to inside) and integrate signals, examine the relationships be- the cell coat, plasma membrane, flattened membratween receptors and effector organelles, and follow the nous “alveoli,” a fibrous “epiplasmic” layer, and varimigration of newly synthesized receptors t o the cell ous filamentous and microtubular structures which surface. Furthermore, electron microscopy can reveal make up the ciliary axonemes and associated “infracilassociations between receptor molecules that may be iature” (Figs. 2-4). These structures are organized to critical to the amplification and transduction of the form “ciliary units,” repetitive units of cortical strucchemical stimulus. ture centered on single or paired cilia and their basal The electron microscope can also provide a wide bodies (Fig. 5). Ciliary units have been extensively range of biochemical and biophysical information characterized by light and electron microscopy in Parthrough its cytochemical, analytical, and ultrahigh amecium (cf. Allen, 1971; Hufnagel, 1969; Iftode et al., resolution capabilities. With a practical resolution ca- 1989; Pitelka, 1965), and Tetrahymena (Allen, 1967). pability close to 0.1 nm, the electron microscope can be Ciliary units are structurally and functionally differused to characterize the morphology and to identify the entiated to participate in different cellular functions, putative binding sites of chemical signals, to compare such as feeding and locomotion, in a manner analogous native and chemically modified molecules, as has been to the differentiation of cell types in a multicellular done for the mating agglutinins of the flagellated pro- organism, a point that was recognized long ago by the tist Chlamydomonas (Crabbendam et al., 1986; Good- Nobel Laureate and expert on ciliate structure, Andre enough et al., 1985; Samson et al., 1987), to detect some Lwoff (1950). Different species of ciliates are charac-

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Fig. 2. Schematic representation of a portion of a pellicle of Paramecium, illustrating the associations between membranes, basal body, kinetodesmal fiber, and microtubules, and based on published observations of a number of investigators. L, R, A, P = animal’s left, right, anterior, and posterior. c.m. = cell membrane; e.1. = epiplasmic layer; i.a.m. = inner alveolar membrane; i.1. = infraciliary lattice; k.f. = kinetodesmal fiber; 0.a.m. = outer alveolar membrane; p. = parasomal sac; t.m.r. = transverse microtubular ribbon; t.p. = transverse plate. (Reproduced from Hufnagel, 1969, with permission of The Rockefeller University Press.)

Fig. 3. The somatic cortex of Cyclidiurn colpodu somewhere in the posterior half of the cell. The observer looks from the anterior toward the posterior end of the cell (which means that the animal’s right side is on the right side of the diagram). At two sites, on the left cilium and over part of the flat slope of one of the pellicular ridges, a small piece of the E-face (E) of the plasma membrane is removed to show the double-stranded ciliary necklace (cn), the ciliary plaques (cp), and a complex pellicular rug (cpr). A system of alveolar sacs (as) is underneath the plasma membrane followed by a thin epiplasmic layer (ep), which is continuous with the terminal plate (tp) of the kinetosome. The cytoplasm in the crest of the pellicular ridges shows one to three longitudinal microtubules (lmt) embedded in the epiplasm, a tube of smooth endoplasmic reticulum (ser), and cistern of rough endoplasmic reticulum (rer). A mitochondrion is usually located underneath the right flat slope of the pellicular ridges in close association with the transverse microtubules (tmt). A non-discharged mucocyst (mc) is shown to the right of the left cilium and a discharged one (dmc) to its left. All membranes except those of the mitochondria and the endoplasmic reticulum are drawn as triple-layered unit membranes. (Reproduced from Bardele, 1983a, with permission of Company of Biologists Ltd.)

locilium” (Ruffolo, 1976a) (see Fig. 6). Emanating from the kinetosomes are a striated kinetodesmal fiber and several types of microtubular ribbons which run parallel to the cell surface (Figs. 2, 3). The plasma membrane near the kinetosomal couplet is invaginated into terized by different structural modifications of their a “parasomal sac,” located a t a fixed angular position ciliary units as well as unique arrangements of ciliary relative to the axis formed by the couplet (Figs. 2-4). units on the cell surface. During cell growth and repro- Since coated vesicles are often associated with it, the duction, an integration of genetic and epigenetic fac- parasomal sac may be a site of receptor-mediated entors regulates cortical unit assembly and differentia- docytosis (Allen, 1976, 1978a). Directly beneath the tion, by mechanisms that are not yet well understood. plasma membrane, over most of the cell surface, is a Ciliary unit structure and arrangement combine to contiguous system of flattened alveoli, which are determine the behavioral properties of a ciliate. Ciliary closely applied to an underlying fibrous layer, the “epiunits are responsible for both reception and response to plasm” (Figs. 2-4). Linked by fibrous connectors to the chemical stimuli. The extent to which a single unit is epiplasm are stationary mitochondria and microtuburesponsible for the entire sequence from reception to lar ribbons. A filamentous layer, the infraciliary latresponse, and the degree of coordination between units, tice, may be found even deeper in the cortex of some are topics which have not yet been addressed, except ciliates (Fig. 7). In Paramecium, this lattice is comfor the coordination of ciliary beating which produces posed of a 23 kDa calcium-binding protein, which imthe “metachronal wave,” a well-known feature of cili- munoelectron microscopy has recently shown to be hoate ciliary behavior. In Paramecium, coordination is mologous to Centrin, the contractile flagellar rootlet thought to be due to viscous-mechanical coupling be- protein of flagellates (Son and Peterson, personal comtween adjacent cilia rather than signals passing from munication). Longitudinal ribbons of microtubules also unit to unit via a cytoplasmic route (Sleigh, 19731, but are present in some species. in oral cilia of another ciliate, Stentor, an internal coCilia ordination process has been proposed to explain meThe numerous cilia (Fig. 8) of the cilioprotist cell are tachronal waves (Sleigh, 1966). Cilia usually occur singly or in pairs, as part of the usually either somatic or oral, and are arranged in linciliary unit. Each cilium emerges from a kinetosome, a ear assemblies whose distributions on the cell surface type of ciliary basal body (Figs. 2-4). In kinetosomal result in species-specific patterns (Fig. 1).Ciliary arpairs (dikinetids), kinetosomes are coupled by a stri- rangements are one of the major criteria used in clasated connective fiber. One or the other kinetosome may sifying ciliate species into different taxonomic groupbe unciliated, or it may have a short, knob-like “condy- ings [Table 1gives the species and genera discussed in

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

Fig. 4. Ultrathin section through basal region of a somatic cilium of Tetrahymena. A = alveolus; BB = basal body; M = mitochondrion; P = coated pit forming at the basal end of the parasomal sac (PS). x 50,000. (Courtesy of M.K. Lyon and L.A. Hufnagel, University of Rhode Island.) Fig. 5. Paramecium tetraurelia: Isolated pellicle, whole mounted, and critical point dried for transmission electron microscopy. The cell cortex is composed of multiple units, each with single or paired basal bodies and attached kinetodesmal fiber (kf). The units are modified in shape, size, number of basal bodies, and kf length, depending on whether they are located close to the oral region (lower right) or more distant from it (upper left). x 8,050.

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Fig. 6. Euplotes eurystomus: Longitudinal section through part of a row of dorsal bristle complexes, showing the condylocilia ( C ) and bristle cilia (B).x 22,560. (Reproduced from Ruffolo, 1976b, with permission of Alan R. Liss, Inc.) Fig. 7. Paramecium multimicronucleatum: A planar view of the infraciliary lattice (IL). Bundles of microfibrils make up this lattice and are not attached to any cell organelle, even though they do occasionally lie next to a basal body (BB). The basal bodies are all sectioned through the level at which kinetodesmal fibers (K) are attached. M = mitochondrion; T = trichocyst tip. x44,350. (Reproduced from Allen, 1971, with permission of The Rockefeller University Press.)

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Fig. 8. Longitudinal thin section through a cilium of Tetruhyme m , paired with a diagram illustrating the locations of fibrous bridges linking the ciliary membrane to microtubules in a typical protozoan cilium. Transitional fibers (T)link the basal bodies to the membrane, microtubule-membranebridges (large arrows)link the doublet microtubules to the membrane, and the microtubule capping structures (small arrows) link the distal ends of the outer and central microtubules to the membrane. (Reproduced from Dentler, 1990, with permission of Plenum Press.)

this article, and the taxa in which they are currently placed, according to a recent revision of the phylum Ciliophora, a subdivision of the kingdom Protista (Lee et al., 1985)l. Some ciliates have relatively simple ciliary patterns, consisting mainly of longitudinal rows of somatic cilia (“kineties”)used primarily for swimming. The most complex patterns may include numerous specialized oral cilia, closely grouped into “membranelles,” which help sweep food particles into the cytostomal region where food vacuoles form, and somatic cilia which are closely arrayed into “cirri,” paintbrush-

shaped appendages used for walking and swimming (Fig. 9). As effector organelles for motility, food capture, or sexual behavior, the cilia are arranged in a variety of patterns representing different behavioral strategies; ciliary patterns may represent different strategies for chemoreception as well. Somatic cilia are directed toward the external environment and so are likely to be the first part of the cell to come in contact with chemical stimuli. Some cilia, like cat’s whiskers, are longer than the rest and project further into the environment. Other cilia are found in feeding structures, such as oral or buccal cavities, where they come in contact with food particles and molecules and may assist in food selection and in directing food particles into the forming food vacuole. The ciliary membranes can represent as much as 50% of total surface area of the ciliate cell, e.g., in Paramecium (Dunlap, 1977; Machemer and Ogura, 1979), and thus provide considerable potential for presenting chemoreceptors to the environment. Cilia with putative chemoreceptive functions exhibit a variety of morphologies. They may be motile, and exhibit a typical ciliary/flagellar morphology (Fig. 8): elongated with an internal cytoskeleton (axoneme) based on a “9(2) + 2’’ arrangement of microtubules, as in the oral cilia of Tetrahymena (Dentler, 1990; Sattler and Staehelin, 1974). A ciliary membrane, continuous with the somatic plasma membrane, sheaths the axoneme and is linked t o it with periodic cross-bridges. Other cilia are ultrastructurally modified, suggesting to some investigators that they serve a chemoreceptive function, although in no case has this been verified (Figs. 1 0 , l l ; Table 2). Characteristics common to these cilia include a distally inflated membrane, distortion of the axoneme, a lack of dynein arms, and an abundance of “particles” when the membrane is studied by freeze-fracture methods, all characteristics shared with putative chemoreceptive cilia of metazoans (cf. Menco, 1980a,b; Schrenk and Bardele, 1987). Such putative chemoreceptive cilia include clavate cilia. Examples are found in and near the vestibulum (a feeding structure) of Balantidium cauiae (Paulin and Krascheninnikow, 1973) and in the oral region of Lagynophrya fusidens (Grain, 1970) and Dileptus cygnus (Grain and Golinska, 19691, locations which suggest a role in feeding. Other putative chemoreceptive cilia of specialized morphology include the scopular cilia of peritrichs (Bardele, 1981), paralabial cilia of the Ophryoscolescidae (Bretschneider, 1962; Schrenk and Bardele, 1987), equatorial cilia of Strombidium (FaureFremiet and Ganier, 19701, and brush cilia of Homalozoon (Leipe and Hausmann, 1989), Spathidium and Bryophylum (Bohatier et al., 1978), Enchelydium (Foissner and Foissner, 19851, and Placus (Grain et al., 1978). Some ciliates have specialized non-motile cilia which may be sensory. Some of these are longer than motile cilia, e.g., the stiff caudal cilia of Paramecium (Machemer and Machemer-Rohnisch, 1983) and Cyclidium (Bardele, 1983a) (Fig. 12; Table 2). Machemer and Machemer-Rohnisch (1983) demonstrated a mechanoreceptive function for the caudal cilium of Paramecium and showed that it transmits a stimulus passively to

ULTRASTRUCTURE AND CHEMORECEPTION I N CILIATES

Fig. 9. Scanning electron micrograph of two Euplotes eurystomus cells, showing the ventral surface (right) containing a scooped out buccal cavity and an array of compound ciliary organelles (cirri and membranelles) and the dorsal surface (left) with its rows of short cilia (bristle-cilia). x 840. (Reproduced from Ruffolo, 1976b, with permission of Alan R. Liss, Inc.)

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Fig. 10. Some putative chemosensory cilia of the ciliophora having variant morphology. A. Balantidiurn caviae: Balloon-shaped clavate cilia in cross-section (left) and longitudinal section (right; the kinetosome is not in the plane of section). A normal cilium, cross-sectioned,

is at upper right. x 90,000. (Reproduced from Paulin and Krascheninnikow, 1973, with permission of Acta Protozoologica.) B Enchelydiurn polynucleaturn: Longitudinal section of cilia of a brush kinety (a kinety is a row of cilia). x 25,000. (Reproduced from Foissner and Foissner, 1985, with permission of Journal of Protozoology.) C: Ophryoscolez purkinjei: Paralabial cilia. Cilia of the top kinety (TK) and anterior cilia of the distal lateral kinety (DLK) show enlarged and abnormal forms (arrowheads) when compared with normal cilia (C). x 4,000. BF = broad fold; ND = nematodesmata. (Reproduced from Schrenk and Bardele, 1987, with permission of J o u r a l ofProtozool0gy.f

receptor channels residing in the nearby cell soma membrane. A chemoreceptive role should also be considered for such cilia, since they project further than other somatic cilia into the environment.

Other non-motile cilia which may have chemoreceptive activity are exceptionally short, e.g., the dorsal bristles and condylocilia of Euplotes, Aspidisca, and other hypotrichous ciliates (Rosati et al., 1987; Ruffolo,

234

L.A. HUFNAGEL

paralabial organelle, possibly in response to changes in pH (Schrenk and Bardele, 1987).

UFig. 11. Schematic drawing of the paralabial organelle of Ophryoscolex purkinjei, located between the inner oral lip (IOL) and the ciliophor (CP), which bears the syncilia (SC). Left of the IOL is the outer oral lip (OOL). The paralabial organelle is composed of a few distal lateral folds (DLF) which alternate with distal lateral kineties (DLK) composed of clavate cilia with 9(2) + 0 axonemes. The central comb-fold (CF) surmounts the top kinety (TK),which gives rise to 9(2) + 2 cilia. Next and posterior to the TK is the wedge-shaped broad fold (BF) accompanied by two proximal lateral kineties (PLK) and the proximal lateral folds (PLF). Underneath the latter, there are several rows of barren basal bodies (BBB). (Reproduced from Schrenk and Bardele, 1987, with permission of Journal ofProtozoology.)

1976b) (Figs. 6,9,22,23;Table 2). These modified cilia are found projecting from pits on the dorsal surface of the cell. A ring of secretory ampules surrounds each pit (cf. Gortz, 1982; see Fig. 22a). Gortz (1982) demonstrated a response of the dorsal bristles to mechanical stimuli (described below), but a chemoreceptive function should not be discounted, since the bristles and accompanying condylocilia project toward the external environment. Putative chemoreceptive cilia of protists may be part of a specialized chemoreceptive organ. For example, in the commensal rumen ciliate Ophryoscolex purkinjii, clavate cilia are found on a complex “paralabial organelle” (Figs. lOC, 111,which contains numerous microtubular “nematodesmata” and microfilament bundles which may assist in retracting and extruding the

Specializations of the Ciliary Membrane Membranes examined by freeze-fracture electron microscopy show distinctive intramembranous particles (IMPs) occurring singly or in ordered assemblies (arrays) in either the external face (E-face) or protoplasmic face (P-face) exposed when membranes are fractured. IMPs are believed to reflect the presence of transmembrane proteins (cf. Branton et al., 1975; Vilmart and Plattner, 1983), which serve as channels for small ions and molecules, receptors for external signals, and anchors for cytoskeletal and extracellular macromolecules. IMPs and IMP arrays with demonstrated roles in active and passive ion movements include those correlated with the presence of a calcium ATPase in the sarcoplasmic reticulum and those found in gap junctions. Based on their positions on the cell surface and their structural organization, some IMPS and IMP arrays have been suggested to have a role in reception or transduction of external chemical stimuli in ciliates (see below). The ciliary membrane has several types of IMPs; some are scattered randomly along the length of the cilium, others are assembled into regular arrays: the “ciliary necklace,’’ “ciliary plaques,” “ciliary rosettes,” “axial rows,” and “oblique rows” (Fig. 13). All may have chemosensory roles, although the evidence is still extremely meager and consists primarily of location on the cell surface, association with external bristles or protrusions, and correlation with sites of binding of calcium or other histochemical properties. In no case has a chemosensory role been clearly demonstrated. The following is a summary description of most of the membrane particle arrays observed so far in ciliates (also refer to Table 3): Ciliary Necklace (Figs. 13,14). A common feature of all somatic and buccal cilia is a loosely organized band of 6-10 nm P- and E-face IMPs, circumscribing the base of the cilium (Bardele, 1981). This ciliary necklace (Gilula and Satir, 1972) is in a region of the ciliary membrane that is pinched in toward the axoneme and attached to it by numerous fibrous bridges (cf. Dute and Kung, 1978; Sattler and Staehelin, 1974) or a dense annulus (Gortz, 1982). In a comparative freeze-fracture study of some 90 different ciliate species, Bardele (1981) found that the necklaces of ciliates always have 2 IMP strands, whereas the necklaces of protistan flagella and the cilia and flagella of metazoa have 3 or more strands of IMPs (Bardele, 1983b; Menco, 1980~). The common feature of two strands in the necklace of ciliates has been corroborated in many other studies of ciliates, including Euplotes (Dallai and Luporini, 1983a,b),Paramecium (Plattner et al., 1973), and Tetrahymena (Wunderlich and Speth, 1972).In frozen-etched preparations of Tetrahymena cilia, Speth and Wunderlich (1972; Wunderlich and Speth, 1972) found that the necklace particles protrude from the external surface of the ciliary membrane, a situation which has also been observed in mammalian olfactory cilia by Menco (1980~). This suggests that necklace proteins may provide binding sites for external signaling

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

235

TABLE 2. Some putative chemoreceptiue cilia of the ciliophora Type of cilium Clavate cilia Scopular cilia Paralabial cilia Equatorial cilia Brush cilia

Caudal cilia Dorsal bristles Condylocilia

Occurrence (examples)

References

Balantidium cuuine, near vestibulum Lugynophrya fusidens, oral region Dileptus cygnus Peritrichs Ophryoscolescidae Strombidium Homalozoon Spathidium, Bryophylium Enchelydium Placus Paramecium Cyclidium Euplotes, Aspidisca Euplotes

Paulin and Krascheninnikow (1973) Grain (1970) Grain and Golinska (1969) Bardele (1981) Bretschneider (1962); Schrenk and Bardele (1987) Faure-Fremiet and Ganier (1970) Leipe and Hausmann, 1989 Bohatier et al. (1978) Foissner and Foissner (1985) Grain et al. (1978) Machemer and Machemer-Riihnisch (1983) Bardele (1983a) Rosati et al. (1987); Ruffolo (1976a,b) Ruffolo (1976a,b)

podida, including Bresslaua, Colpoda, and Tillina (Bardele, 1981, 1983b). These plaques are orthogonal P-face arrays of large (6-12 nm) IMPs, consisting of 3 longitudinal rows of 2-25 particles. In Cyclidium, Bardele (1983a) found that the non-motile sensory caudal cilium did not have plaques, although an increased density of large IMPs was noted in the “plaque area.” Oral cilia of CycEidium had P- or E-face plaques of somewhat different particle arrangement than somatic cilia. In Tetrahymena, some oral cilia have plaques while others do not (Sattler and Staehelin, 1974). Where present, there are usually nine plaques, corresponding to the nine outer doublet microtubules, arranged around the base of the cilium, just distal to the ciliary necklace. Thin sections reveal that the ciliary membrane balloons out from the axoneme in the plaque region. The resulting space contains dense material linking membrane and axoneme (Dute and Kung, 1978; Wunderlich and Speth, 1972) (see Figs. 4, 8). In Tetrahymena, the ciliary plaques are longest on the cilia closest to the anterior end of the cell and reduced in length on posterior cilia (Hufnagel, unpublished). Ciliary plaques selectively bind external ligands or are correlated with relevant enzyme activities. The Fig. 12. A drawing of Cyclidium glaucoma, showing the ventral surface dominated by the oral apparatus. The somatic cilia are shown membrane surface in the ciliary plaque region of Parin resting position. cc = stiff caudal cilium. (Reproduced from amecium has a concentration of anionic sites, demonBardele, 1983a, with permission of Company of Biologists Ltd.) strated ultrastructurally by the binding of polycationic ferritin (Dute and Kung, 1978). Calcium affinity sites, molecules; however, there is no direct evidence for this perhaps representing calcium channels, also have been in ciliates. Unlike the ciliary necklace of rabbit oviduct localized to the plaque region of Paramecium (Plattner, (Anderson and Hein, 1977), the necklace of Parame- 1975) (Fig. 15),by the technique of Oschman and Wall cium was not correlated with external binding sites for (1973), and the OsO, oxalate method of Constantin et cationic ferritin (Dute and Kung, 1978), and this au- al. (1965). However, calcium affinity sites also occur in thor was not able to uncover any other published evi- ciliates such as Spirostomum which lack ciliary dence for specific ligand binding a t the ciliary necklace plaques (Bardele, 1983a). A calcium-ATPase has been correlated with plaques in Tetrahymena (Baugh et al., of ciliates. Ciliary Plaques (Figs. 13,14,16). A second type of 1976). Ciliary Rosettes (Figs. 13,16). Ring-like arrays of IMP array, the plaque (Wunderlich and Speth, 1972) or ciliary granule plaque (Plattner, 1975; Plattner et al., about 11 IMPs, called ciliary rosettes, have also been 1973; Satir et al., 1976), is found on the motile somatic observed in ciliary membranes of some free-living hycilia of Paramecium (order Peniculida) and related cil- menostome ciliates of the genus Frontonia Bardele, iates belonging to the orders Hymenostomatida and 1980a, 1981). Similar rings of 9-12 particles, about 7 Scuticociliatida, and also in some members of the Col- nm in diameter, have been observed in the P-face of the

i“

236

L.A. HUFNAGEL

TABLE 3. Ciliary membrane particle arrays o f the ciliophora, having possible chemoreceptive functions’

Array Necklace Plaques

Rosettes Longitudinal rows

Oblique arrays

Distal arrays Basal arrays

Small rosettes Rosette-like arrays

Patches

Features Double row around base of cilium (Figs. 13, 14) 3 longitudinal rows of 2-25 particles near base of cilium distal to necklace (Figs. 13, 14, 16) Ring-like array of -11 IMPs (Figs. 13, 16) Parallel to axis of cilium; single or paired rows; particles aligned or in zig-zag arrangement (Figs. 13, 17, 21B) Numerous obliquely oriented rows, closely spaced (Figs. 18, 20, 21A, 34A) Loosely organized short rows or doublet rows (Fig. 22A,B) Plate-like, associated with “lasiosome” network near base of cilium (Figs. 22A, C) Ring of 5-6 particles (Fig. 40) A few IMPs on membrane-protrusions over underlying dense bodies (Fig. 41B) Up to 50 IMPs in a loose cluster (Fie. 41A)

‘E = external; IMP = intramembranousparticle; P ‘Branton et al. (1975).

=

Membrane face’ PandE

Particle size (nm) 6-10

P

6-12

P

?

P

Occurrence All cilia Motile cilia of some orders

References Gilula and Satir (1972); Wunderlich and Speth (1972) Bardele (1981, 1983b); Plattner et al. (1973)

Frontoniu

Bardele (1980a, 1981)

Various

Oral cilia of Tetrahymena, Glaucoma; somatic and oral cilia of other ciliates

Bardele (1980a, 1981); Montesano et al. (1981); Sattler and Staehelin (1974); Hufnagel (unpublished)

P

Various

Oral cilia of Tetrahymena; putative chemoreceptive cilia of other ciliates

P

4-5

Euplotes, bristle-cilia

Bardele (1980a,b, 1981); Montesano et al. 11981); Sattler and Staehelin (1974); Hufnagel (unpublished) Dallai and Luporini (1983a,b)

P

4-5

Euplotes, bristle-cilia

Dallai and Luporini (1983a,b)

P

3-4

Hufnagel (1979, 1984)

P

?

Tetrahymena, anterior cilia of mating and mating-reactive cells Euplotes, mating-reactive

P

?

Euplotes, mating-reactive

Dallai and Luporini (1983a); LuDorini and Dallai (1981)

Dallai and Luporini (1983a); Luporini and Dallai (1981)

protoplasmic.

Oblique Arrays (Figs. 18, 19). Bardele (1980a, flagellar membranes of the flagellated protist Tritrichomonas foetus (Benchimol et al., 19821, a urogenital 1981) described prominent arrays consisting of thoupathogen of cattle. These flagella are usually inactive sands of IMPS in closely spaced, obliquely oriented except for occasional beating in response to contact rows on cilia of Spirostomum and Tracheloraphis, rewith obstacles (Amos et al., 1979). Benchimol et al. lated ciliates which are highly contractile and have (1982)suggested that these flagella have a role in sens- “thigmotactic” (adhesive) cilia. Very similar arrays ing the environment. (Another type of ciliary rosette, have been seen in the somatic plasma membrane of the small ciliary rosette observed only in mating Tet- Spirostomum (Bardele, 1980b, 1981) and the related contractile ciliate Stentor (Bardele, 1980b, 1981; rahymena, is discussed later.) Longitudinal Rows (Figs. 13,17). Single or paired Hufnagel and De Terra, unpublished) (see Fig. 19), but axial rows of IMPs have been described on somatic and were not found on related heterotrichs which are nonoral cilia of a few species, including Pseudomicrothorax contractile (Bardele, 1981). Bardele (1981) coined the and some members of the order Entodiniomorphida term orthogonal arrays for these IMP assemblies of (Bardele, 1980a, 1981),and on certain oral cilia of Tet- contractile ciliates. Obliquely oriented arrays observed rahymena pyriformis (Sattler and Staehelin, 1974), T. on oral cilia of the non-contractile ciliates Tetrahymena thermophila (Hufnagel, unpublished), and Glaucoma pyriformis (Sattler and Staehelin, 1974), T. thermoferox (Montesano et al., 1981). Bardele (1981) observed phila (Hufnagel, unpublished), and Glaucoma ferox in thin sections fibrous material linking adjacent cilia (Montesano et al., 1981) are associated with external and proposed that the IMPSof longitudinal ciliary rows fibrous extensions (see below) and may be unrelated to may in some cases serve as anchoring sites for such the orthogonal type of array: interciliary fibrils. These fibrils may serve more than a Specialized Arrays of Putative Sensory Cilia passive role in mechanically linking the cilia together (Fig. 20). The clavate cilia of Loxophyllum meleagris and a role as transmission wires for both mechanical and Litonotus pictus, scopular cilia of Vorticella sp., and chemical signals should be considered. paralabial cilia of Ophryoscolex sp., and equatorial cilia

237

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

TABLE 4. Somatic and oral membrane particle arrays of the ciliophora, having possible chemoreceptive functions' Membrane face' -

Particle size (nm)

4-25 IMP rows spaced 25-30 nm part; anterior locations (Figs. 24, 25, 27)

P

11-15; composed of an 8-9 nm IMP and two 5-6 nm IMPs

Three rows of IMPs spaced 25 nm apart, anterior half of cell (Figs. 24, 28A) Similar to simple rugs but flanked on either side by two rows of smaller IMPs (Figs. 24, 28B,C) Ring of 8-10 IMPs, sometimes around a central IMP; sometimes surrounded by larger particle rings; associated with docked extrusome (Figs. 38C, 39B) Ring-shaped field of IMPs associated with unciliated basal bodies; mav be uroeenitor of ciliary Geckulace (Figs. 28A, 29) Double rows of IMPs; spaced 7.5-8.0 nm apart; associated with underlying microtubules or alveolar margins; anterior location (Figs. 25,27,31A) Long, narrow fields containing short, diagonal rows of 3-4 IMPs (Fig. 31B) Ovoid track of IMPS, surrounding fixed defecation site (cytoproct) (Figs. 24, 30)

P

10

P

Single rows of large IMPs spaced -33.5 nm apart, along flanks of ribs (Fig. 35) Linear grouping of small IMPs, spaced 13.5 nm apart, in grooves between ribs (Fie. 35)

Array Somatic Plate-like arrays

Simple pellicular rugs Complex pellicular rugs Fusion rosettes

Fairy rings

Paired linear arrays

Tetragonal arrays

Cytoproct arrays

Oral Oral rib arrays I

Oral rib arrays I1

Features

-

Occurrence

References

Many species, including Paramecium, Cyclidium, Tetrahymena Cyclidium

Allen (1976, 1978a,b); Bardele (1983a); Hufnagel (1981a,b); Preston and Newman (1986)

12,ll

Cyclidium

Bardele (1983a)

10-15

Tetrahymena Paramecium Euplotes Eufolliculina

Hufnagel (1981b); Wunderlich and Speth (1972) Plattner et al. (1973) Dallai and Luporini (1981) Mulisch et al. (1989)

Bardele (1983a)

P

6-18

Tetrahymena, Cyclidium

Bardele (1983a); Hufnagel (1983); Satir et al. (1972a)

P

6

Tetrahymena, Paramecium

Allen (1978b); Hufnagel (1981b)

P

10

Tetrahymena, Eufolliculina

Hufnagel (1981b); Mulisch et al. (1989)

E

?

Paramecium, Tetrahymena, Cyclidium

Allen (1978b); Bardele (1983a); Hufnagel (1981b)

P

?

Tetrahymena

P

?

Tetrahymena

Hufnagel (unpublished) Sattler and Staehelin (1979); Wunderlich and Speth (1972); Sattler and Staehelin (1979); Wunderlich and Speth (1972); Hufnagel (unwblished)

Y

'Arrays most likely to have a chemoreceptivefunction are placed first in the list. E 'Branton et al. (1975).

of Strombidium are heavily studded with IMPs organized into circumferential rows (Bardele, 1981). Scopular cilia of peritrichs like Vorticella are believed to serve a sensory function related to selection of a substrate attachment site for the free-swimming motile swarmer. The clavate cilia of Loxophyllum and Litonotus are present on the dorsal side of these bottomdwelling ciliates and may also have a role in substrate selection. Comparative Study of Ciliary Membranes in Euplotes (Figs. 21, 22). In ciliates of the order Euplo-

=

external; IMP = intramembranousparticle; P = protoplasmic

tida, which are presumed to be evolutionarily advanced, there are three distinct types of cilia (see Fig. 9), which affords the possibility of interesting studies of comparative membrane morphology within a single cell. Motile cilia are ventrally located and grouped either in cirri used for walking and swimming or in a prominent band of membranelles used in feeding. Short, dorsally located bristle-like cilia are a common feature of the Euplotida. These have been shown to tilt in response to fluid flow (Gijrtz,1982), but are otherwise non-motile. Ruffolo (1982) speculated on a possi-

238

L.A. HUFNAGEL

ing Euplotes were also examined and have two new types of specializations, described later. Dallai and Luporini also found that Euplotes dorsal bristle-cilia have three unique arrays. The basal array, present in all three species near the base of the bristlecilium, consists of 4-5 nm particles in an orderly platelike arrangement of parallel rows spaced 12 nm apart (Fig. 22A,C) and appears to be associated with the “lasiosome” network, a regular arrangement of electrondense granules lying between the anterior side of the axoneme and the ciliary membrane ( G r t z , 1982; Ruffolo, 1976a) (see Fig. 23). It is found only in members of the genus Euplotes and the closely related ciliate Certesiu (Wicklow, 1983). Both Ruffolo (1982) and Gortz (1982) proposed that the lasiosome complex has a role in mechanosensory transduction, but a role in transduction of chemical stimuli seems equally likely; the bristle-cilia are very short, lie partly protected in pits on the dorsal surface, and thus seem ill-suited for mechanoreception. A second bristle array was found in only one species, E . ruikoui (Dallai and Luporini, 1983a1, and consists of a disordered assembly of longitudinal particle rows, occupying a major portion of the

Fig. 13. Diagram of a cilium, showing how the freeze-fracture technique splits the ciliary membrane through the middle of its hydrophobic liquid bilayer and reveals several distinct arrays of intramembranous particles on the protoplasmic (P)lamella, leaving corresponding pits on the external (E) lamella. The most conspicuous particle arrays are the double-stranded ciliary necklace, the ciliary plaques, the ciliary rosette, and the longitudinal rows. (Adapted from Bardele, 1981,with permission of Elsevier Scientific Publishers Ireland Ltd.)

ble role of these “bristle-cilia” in photo- or mechanoreception, but a chemoreceptive role should also be considered, since these cilia are in a good location to sense chemicals flowing past the cell. It is not clear what purpose is served by a change in tilt angle. One possibility is that tilting is required for mechanoreception to occur, but another is that tilting of the bristles helps to expose chemoreceptors located at the ciliary base to molecular signals flowing past the cell. Parenthetically, the experimental set-up described by Gortz (1982) might provide an excellent one for testing the effects of iontophoretically applied chemical stimulants on the tilt response, or the effects of the tilt response on chemoreception. Dallai and Luporini (1983a,b; Luporini and Dallai, 1981) conducted a comprehensive comparison of the membranes of these three types of cilia, using freezefracture electron microscopy, in three species of marine Euplotes. All cilia were found to have the typical double-stranded ciliary necklace. In cilia of cirri and membranelles, numerous additional transverse rows of IMPs were seen, where ciliary plaques would be found in Hymenostome ciliates (Fig. 21A). In addition, single or paired longitudinal rows were found, usually one per cilium, on cirral, membranellar, or dorsal bristle-cilia (Fig. 21B). These extend for varying lengths along the ciliary shaft and have particles that are regularly aligned or in a zig-zag arrangement. The cilia of mat-

Fig. 14. Cilium of Tetruhymena, freeze-fractured to reveal basal arrays of IMPs. A: P-face of the ciliary membrane, showing irregularly spaced particles of the ciliary necklace below the larger tetragonally arranged IMPS of the ciliary plaques. More distal regions of the ciliary membrane are characterized by numerous randomly dispersed IMPs. x 71,100.B E-face of the ciliary membrane, showing the double-stranded ciliary necklace. (For proper viewing, turn page 180”.)x 75,400.(Reproduced from Hufnagel, 1983,with permission of Company of Biologists Ltd.) Fig. 15. Paramecium tetruurelia: Electron micrograph of a cell exposed to 10 mM calcium and subsequently fixed in glutaraldehyde only. Electron-dense deposits occur on the basal portion of cilia, where the ciliary plaques are located. Stain is closely attached to the inside of the ciliary membrane. Some deposits are found in more apical regions of cilia. x 36,200.(Reproduced from Plattner, 1975,with permission of Company of Biologists Ltd.) Fig. 16. Six examples of ciliary plaques and rosettes in Frontonia leucus. x 45,000.(Reproduced from Bardele, 1981,with permission of Elsevier Scientific Publishers Ireland LM.) Fig. 17. Examples of longitudinal rows of IMPS on cilia. A Syncilia from an entodiniomorph ciliate (probably Ophryoscoler sp.) show long regular particle rows along almost the entire lengths of the cilia. x 45,000. (Reproduced from Bardele, 1981, with permission of Elsevier Scientific Publishers Ireland LM.)B: Cilium from the outer row of the third membranelle of Tetruhymena pyriformis. Three rows of particles are found on the P-face, spaced about 81-108 nm apart, suggesting that the particles could be associated with elements of the underlying microtubule doublets. x 84,850.(Reproduced from Sattler and Staehelin, 1974,with permission of The Rockefeller University Press.) Fig. 18. Oblique arrays. A Tracheloruphis sp.: Orthogonal E-face array of the ciliary membrane in non-chemically fixed specimen of this sand ciliate. X 60,000. (Reproduced from Bardele, 1981,with permission of Elsevier Scientific Publishers Ireland Ltd.)B Spirostomum intermedium: A similar orthogonal E-face array of the ciliary membrane of an unfixed specimen. In cells fixed with glutaraldehyde, the particles stay with the P-face. ~60,000. (Reproduced from Bardele, 1980a,with permission of Walter de Gruyter & Co.) Fig. 19. Stentor: An orthogonal array of unknown location. x 62,500.

ULTRASTRUCTUREAND CHEMORECEFTION IN CILIATES

Figs. 14-19.

239

240

L.A. HUFNAGEL

Fig. 20. IMP arrays of putative chemosensory cilia. A: Loxophyllum meleugris, anterior segment of a clavate cilium. x 45,000. B: Ophryoscolex sp.: Cross-fracture through the “fir-cone”-shaped paralabial organ. It is composed of several parallel folds (pf) containing numerous microtubules. Between the pellicular folds there are rows of short “labial cilia” (lc). They have a larger diameter than ordinary cilia and bear numerous twisted rows of particles. Some cilia (arrowhead) show particle arrays which resemble those seen in clavate cilia.

x 30,000. (Reproduced from Bardele, 1981, with permission of Elsevier Scientific Publishers Ireland Ltd.)

Fig. 21. Ciliary IMP arrays in Euplotes. A: E . ruriseta: Transverse rows of IMPS in the proximal region of cirral cilia. x 56,200. B: E . crussus: Longitudinal rows of IMPS on membranellar cilia of mating cells. x 45,200. (Reproduced from Dallai and Luporini, 1983a, with permission of Journal of Submicroscopic Cytology and Pathology.)

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

241

Fig. 22. Ciliary IMP arrays in Euplotes. A Bristle-cilium bearing the basal plate-like array (visible in detail in B) and the apical array (visible in detail in C). Ciliary ampules (CA) surround the pit containing the bristle-cilium. A: x 46,900; B, C: X 75,000. (Reproduced from Dallai and Luporini, 1983a, with permission of Journal of Submicroscopic Cytology and Pathology.)

Fig. 23. Euplotes eurystomus: Approximately longitudinal section through a dorsal bristle complex, showing the couplet kinetosomes, bristle-cilium (B) containing lasiosomes (L), and condylocilium (arrowhead). The complex lies in a pit, and a portion of the pit wall forms a shelf (SH) over the condylocilium. x 63,360. (Reproduced from Ruffolo, 1976a, with permission of Alan R. Liss, Inc.)

ciliary shaft and apparently connected to the axoneme by cross-bridges seen in thin sections. All species of Euplotes examined by Dallai and Luporini (1983b) also have distal arrays on the ends of the bristle-cilia (Fig. 22A,B). These are loosely disordered arrays of 4-5 nm particles, in short rows or numerous doublet rows. The underlying organization of the distal part of the cilium was not studied in detail. However, in Tetrahymena and other ciliates a terminal complex of granular and fibrous elements, linking the ciliary membrane to microtubules of the axoneme and named the ciliary cap, has been described (Dentler, 1980,

1984) (Fig. 8). Such internal membrane-cytoskeletal linkers, as well as external hairs, seem to be common features of the tips of cilia and flagella (Dentler, 1981), and may mediate the regulation of axonemal function in response to external chemical signals. Membrane-Axonemal Bridges. Most, if not all, ciliary membrane arrays are linked to the axoneme by fibrous cross-bridges, consistent with the possibility of sensory transduction roles for both IMPS and bridges (cf. Sattler and Staehelin, 1974). Additional linkages occur a t intervals of about 220 nm in Tetrahymena (Dentler et al., 1980) (Fig. 8). These bridges may be

242

L.A. HUFNAGEL

correlated with the longitudinal rows of IMPs such as those of oral cilia (Sattler and Staehelin, 1974; Hufnagel, unpublished), or with the more randomly distributed particles found on somatic cilia. Efforts to show a role for these bridges in regulation of ciliary motility have been suggestive but not conclusive (Dentler et al., 1980). The increased density of random IMPs found on putative sensory cilia suggests more frequent linkage of the membrane to the axoneme in these cilia (Bardele, 1981).

Specializations of the Somatic Plasma Membrane A variety of ordered IMP assemblies are found in the somatic cell membrane of ciliates. Some occur in many different species while others are species or genus specific. Often, these arrays occur at sites that suggest a chemoreceptive function, e.g., near the anterior end of the cell, feeding organelles, or cilia (Figs. 24-26). Somatic IMP arrays with potential chemoreceptive functions are described below (also refer to Table 4). Plate-Like Arrays (Figs. 24-27A,B). First described in Paramecium multimicronucleatum by Allen (l976,1978a,b; for correction of species name see Allen, 1988), who called them rectilinear plates, these are common to many ciliate species (Bardele, 1983a), including Tetrahymena thermophila (Hufnagel, 1981a,b), Cyclidium glaucoma (Bardele, 1983a1, and P. tetraurelia (Preston and Newman, 1986). In P. tetraurelia, these arrays are numerous and lie mainly on the flanks of pellicular ridges in the left dorsoventral region anterior to the buccal cavity and contractile vacuole pore (Fig. 26). In Tetrahymena, they lie on the ventral interkinetal ridges anterior to the oral cavity (Fig. 25). In Cyclidium, there are four larger and four smaller plates, all in fixed positions near the anterior end or in the buccal cavity (Fig. 24). Particle plates vary widely in size; they can be up to 2 p,m in length and occupy an area up to 1.2 pm2 (Hufnagel, 1981a,b). However, their structure is reCAWLL ClLluY VENTRAL SIDE OF markably constant between ciliate species (Fig. 27A). CYCLlDlUM GLAUCOMA They are composed of between 4 and 25 particle rows spaced 25-30 nm apart. Each row consists of closely spaced units about 11-15 nm in diameter. When Fig. 24. Cyclidium glaucoma: Drawing summarizing distribution viewed a t high resolution, in Cyclidium and Tetrahy- of membrane arrays in relation to somatic and buccal ciliature and menu, the particle rows appear to be tripartite, with other ventral surface landmarks. Ciliated kinetosomes and those that can potentially bear cilia are shown as large open circles. The black each larger particle (8-9 nm diameter in Cyclidium) dots near them represent parasomal sacs. Plate-like arrays (PP),simflanked by two smaller ones (5-6 nm diameter in Cy- ple pellicular rugs, and complex (luxurious) pellicular rugs are shown clidium). as regular patterns of dots; except for the plates, they are not drawn A chemosensory function for plate-like arrays has to scale. Note that the plates (PP,_,; PP,-,) are restricted to the anof the cell and the buccal cavity. Their location and orienbeen repeatedly proposed because of their anterior-ven- terior pole are fixed. Simple pellicular rugs occur in the anterior half of tral and buccal locations (Allen, 1978b;Bardele, 1983a; tation the cell, while complex rugs are associated with monokinetids in the Hufnagel, 1981a,b; Preston and Newman, 1986). This posterior half of the cell. The border between the simple and complex view is supported by the finding in Tetrahymena rugs marks the fission zone during vegetative reproduction. A cytoarray is also shown, posterior to the buccal region. PK1-3, buc(Hufnagel, 1981a) and P. tetraurelia (Preston and New- proct membranelles. (Reproduced from Bardele, 1983a, with permission man, 1986) that portions of these structures protrude cal of Company of Biologists Ltd.) from the external surface of the cell (Fig. 27B). Platelike arrays may be closely associated with the underlying outer and inner alveolar membranes (Hufnagel, suggestive of a physiological function related to 1981b; Preston and Newman, 1986), providing a direct chemoreception. structural link between the external surface, the alveRecently, Lutke and Plattner (1986) reported that olar lumen, and the cytosol, an arrangement highly the lectin wheat germ agglutinin (WGA), which binds

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/

Fig. 25. Tetrahymena thermophila: Diagram illustrating apparent distribution of particle arrays of P-face of plasma membrane relative to surface topography. Note that all arrays are anterior to or within the oral cavity. C = position of cilia; P = paired arrays; PL = platelike arrays; T = tetragonal arrays. (Reproduced from Hufnagel, 1981b, with permission of Journal ofProtozoology.)

Fig. 26. Paramecium tetraurelia. Localization of plate-like IMP

to GlcNAc residues, labels longitudinal rows of regular arrays on the ventral (left) and dorsal (right) surfaces. Each point cell surface fields in the anteroventral region of P. tet- represents an array whose location was accurately determined from of identified portions of the plasma membrane. The figure is raurelia. This region is where plate-like arrays are con- replicas a cumulative representation of results from a number of different centrated, where, as Lutke and Plattner (1986) note, replicas and is not representative of the number of arrays possessed various researchers have demonstrated the presence of by any one cell. Note that few arrays are found posterior to the antesensory receptors and ion channels related to sensory rior contractile vacuole pore (acvp). (Reproduced from Preston and reception (see below) and where somatic membranes Newman, 1986, with permission of Company of Biologists Ltd.) make junctional contact during cell pairing accompanying sexual reproduction. Simple Pellicular Rugs (Figs. 24, 28A). In Cy- (Bardele, 1983a). They differ from the latter in having clidium glaucoma, Bardele (1983a) discovered arrays two different sizes of particles: three rows of 12 nm of 10 nm particles oriented in three parallel rows particles are flanked on each side by two more closely spaced 25 nm apart. Each array, which he called a spaced rows of 11 nm particles. Again, similar arrays simple pellicular rug, is between 0.2 and 0.6 p,m long. have not been reported in other ciliates. Like the simArrays are found only in the anterior half of the cell ple pellicular rugs, these may have a chemoreceptor and are closely associated with the bases of cilia, but function related to the activity of nearby cilia or paraare oriented randomly with respect to the anterior-pos- soma1 sacs. Fairy Rings (Figs. 28A, 29). First described in Tetterior axis of the cell or the cilia themselves. Bardele (1983a) called attention to the morphological similar- rahymena by Satir et al. (1972a))these have also been ity of these rugs to ciliary plaques, but found no struc- observed in Cyclidium (Bardele, 1983a) and presumtural evidence that the rugs and plaques are function- ably occur in all ciliates. The name was coined by ally interchangeable. Similar arrays have not yet been Hufnagel (1979)) who also analyzed their behavior in observed in other ciliates. Because of their locations, relation to ciliogenesis (Hufnagel, 1983). Fairy rings these arrays are candidates for a chemoreceptive func- consist of doughnut-shaped fields of particles, irregution related t o the nearby cilia or parasomal sacs, al- larly arranged or in clusters, located in the plasma though a mechanoreceptor function was suggested by membrane just above the unciliated basal bodies of dikinetids. While they may be progenitors of the ciliary Bardele (1983a). Complex Pellicular Rugs (Figs.24,28B,C). These necklace (Satir et al., 1972a), fairy rings may also have arrays occur in the posterior half of Cyclidium glau- a receptor function related to basal body behavior or coma, in locations similar to the simple pellicular rugs morphogenesis.

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Fig. 27. Plate-like arrays of Tetrahymena thermophila. A A platelike array (pl) and nearby paired linear array (p) of T. thermophila, shown in a P-face replica of the plasma membrane. A portion of the underlying E-face of the outer alveolar membrane (oam) is also revealed. x 100,000.B: A replica of the external surface of the cell, near the anterior end, reveals a domain of ridges, surrounded by remnants of extruded mucus. x 105,700. (B: Reproduced from Hufnagel, 1981a, with permission of Academic Press.) Fig. 28. Particle “rugs,” fairy rings, and other features of Cyclidium. A Posterior end of the anterior part of kinety no. 10, showing dikinetids with the posterior kinetosomes ciliated (c). The anterior non-ciliated kinetosomes are identified by the presence of fairy rings (fr). Particles of the fairy rings often come in groups of four. Simple pellicular rugs (spr) lie close to the ciliary row, are variable in length,

and are oriented randomly. Arrowheads mark the parasomal sac openings. x 39,000. B, C: The caudal cilium (c) emerges from a small depression a t the posterior end of the cell; it is accompanied by two parasomal sacs and one to five complex pellicular rugs (cpr). Complex rugs may be relatively simple, as in C, or compound, as in B. m = recently discharged mucocyst. x 50,000. (Reproduced from Bardele, 1983a, with permission of Company of Biologists Ltd.) Fig. 29. Tetrahymena thermophila, E-face of the plasma membrane, looking from inside the cell toward the exterior. A fairy ring (arrowhead) is located anterior to the broken shaft of a cilium (c). Just anterior to the fairy ring is a parasomal sac (p) and a similar one lies anterior to the cilium. x 41,000. (Reproduced from Hufnagel, 1983, with permission of Company of Biologists Ltd.)

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

Fig. 30. Cytoproct array of Tetrahymena, E-face of the plasma membrane (PM) and P-face of the outer alveolar membrane (OAM). Elevated region in center of particle track may be site of membrane fusion during defecation (arrowhead). x 36,300. (Reproduced from Hufnagel, 1981b, with permission of Journal ofProtozoology.) Fig. 31. A: Paired linear arrays (p) of Tetrahymena thermophila in the P-face of the plasma membrane. These lie along the crest of anterior cortical ridges. A small portion of a tetragonal array is also present (arrowhead) in a valley between two ridges. x 83,800. B: Tetragonal arrays (t) of T. thermophila, located in the grooves between anterior cortical ridges. P-face of the plasma membrane. x 83,800. (Reproduced from Hufnagel, 1981, with permission of Journal of Protozoology.)

Cytoproct Arrays (Figs. 24, 30). In ciliates, an ovoid track of E-face IMPS surrounds the region near the posterior end of the cell where food vacuoles dock t o release waste materials. This structure has been described in Tetrahymena (Hufnagel, 1981b), Parame-

245

Fig. 32. Cell coat revealed by cytochemical staining. A Litonotus duplostriatus: Somatic surface coat (sc) revealed after fixation in glutaraldehyde-osmium tetroxide. Note filamentous projections. a = alveoli; m = mucocysts. ~ 4 9 , 7 5 0 .(Reproduced from Hausmann and Mocikat, 1976, with permission of European Journal of Cell Biology.) B: L.dupplostriatus:Surface coat (arrowhead) after alcian blue staining. The mucocyst (m) membrane remains unstained. x 50,000. (Reproduced from Hausmann and Mocikat, 1976, with permission of European Journal of Cell Biology.) C: Paramecium caudatum: Ciliary membrane stained with ruthenium red. x 60,000. (Reproduced from Watanabe, 1990, with permission of Plenum Press.)

cium (Allen, 1978b), and Cyclidium (Bardele, 1983a), and is thought to anchor the filaments which connect the borders of underlying alveoli to the plasma membrane. It surrounds the particle-free membrane area which fuses with the food vacuole to create an opening

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through which defecation can occur. It is not known if the cytoproct can open in response to external signals, but in Tetrahymena external application of the lectin Concanavalin A (Con A) interferes with membrane events a t the cytoproct during defecation (Scott and Hufnagel, 1983). Paired Linear Arrays (Figs. 25,31A). Anterior to the oral cavity of Tetrahymena, the cell surface is thrown into deep folds, forming a series of ridges and grooves. Along the crests of the ridges, there are paired rows of small (6 nm) IMPs in the P-face of the plasma membrane (Hufnagel, 1981b). A similar array was observed within the oral cavity. These arrays may coincide with the boundaries of underlying alveoli and could be purely anchoring proteins which applique the edges of the alveoli to the plasma membrane. However, they do not occur in most locations where alveolar boundaries underlie the plasma membrane. Their localized distribution mainly anterior to or within the oral cavity makes these arrays candidates for a role in sensory reception. Similar arrays occur in Paramecium where microtubules underlie the plasma membrane (Allen, 1978b). At alveolar borders in Paramecium, Allen (197813) instead found broad bands of IMPs composed of many particle rows which parallel the long axis of the band. Tetraponal Arrays (Figs. 25, 31A3). In Tetrahymena, in the bottoms-of the anterior cortical grooves, Hufnagel (1981b) observed long, narrow fields of tetragonally arranged 10 nm particles with a center-to-center spacing of 11 nm. The IMPs within the fields are aligned in short diagonal rows of three to four particles. Because of their locations within the grooves, these arrays have been seen only rarely. Eufolliculina has small rectangular particle arrays which show some similarity to the plate-like arrays of other ciliates (Mulisch et al., 1989). However, the size and spacing of particles in these arrays suggest a homology with the tetragonal arrays of Tetrahymena. The Eufolliculina arrays consist of parallel rows of 410 particles spaced about 10 nm apart. The arrays become more numerous and increase in size during formation of the lorica, a secreted tube housing the cell, and are extremely abundant in the trophic cell where they sometimes are found above borders between adjacent cortical alveoli. A signal reception role for these arrays was considered to be unlikely (Mulisch et al., 1989). However, they are located in the plasma membrane above postciliary microtubular fibers, which are suspected of having a role in elongation of the cell following contraction (Sleigh, 1981). This raises the possibility that they may harbor transmembrane channels that regulate motile activity of the cell. Alternatively, they may be membrane anchors for fibers connecting the plasma membrane to underlying membranes or microtubules. The so-called zipper-line of the plasma membrane of hemipteran spermatozoa has a strong resemblance to these tetragonal arrays of ciliates and is associated with short spikes which connect the plasma membrane and one of the two mitochondria1 derivatives characteristic of these sperm (Dallai and Afzelius, 1982).

Cell Coat (Glycocalyx) The external surface of the ciliate cell body and cilia is covered by a surface “coat” or “glycocalyx,” a continuous and more or less flocculent layer which is quite thin in some species and much thicker in others (cf. Hausmann and Mocikat, 1976; Nilsson and Behnke, 1971; Wyroba and Przelecka, 1973) (see Fig. 32A-C). This layer is best revealed a t the ultrastructural level when the cells are pretreated with, or fixed in the presence of, chemicals that stabilize glycoproteins or polysaccharides, such as tannic acid (Hausmann, 1979; Hufnagel, unpublished), alcian blue (Hausmann and Mocikat, 1976) (Fig. 32B), ruthenium red (Henk, 1979; Watanabe, 1981; Wyroba and Przelecka, 1973; Hufnagel, unpublished) (Fig. 3 2 0 , or anionic or cationic ferritin (Nilsson and Van Deurs, 1983; Hufnagel, unpublished) (see Fig. 34B). In general, little is known about the molecular composition or organization of the cell coat, although in Paramecium and Tetrahymena it is thought to contain the major cell surface antigen, the so-called “immobilization antigen” (IA) (Bolivar and Guiard-Maffia, 1989; Hansma and Kung, 1975; Wyroba, 1977, 1980). Immunocytochemical studies suggest that the IA of the cell coat of Tetrahymena originates from secretory organelles known as “mucocysts” (Bolivar and Guiard-Maffia, 19891, and it is reasonable to suppose that other components of the ciliate cell coat also arise from mucocysts or other extrusive organelles. Because of its location and the likely presence of glycoconjugates, the cell coat almost certainly has a role in chemoreception. Recently, the role of secreted mucus in perireceptor events accompanying chemosensory processes in vertebrate olfactory mucosa has received attention (Carr et al., 1990); an analogy can be drawn between this system and the ciliate cell coat. In vertebrates, access of odorants to receptors is thought to be mediated by so-called “odorant binding proteins” (OBPs) located in the perireceptor mucus layer (Snyder et al., 1988). A similar situation exists in arthropod antenna1 sensilla, in which odorants such as pheromones must pass through an aqueous lymph to reach receptors located on ciliated dendrites of olfactory neurons (Vogt et al., 1988). These odorants are thought to be transported to their receptors by “pheromone binding proteins” (PBPs) (summarized in Carr et al., 1990). OBPs and PBPs could act to up- or down-regulate the concentration of odorants in the vicinity of receptors or they could participate directly in the odorant-receptor interaction (Carr et al., 1990). The ciliate cell coat may have many of the general chemical properties of mucus and lymph, such as an abundance of polyionic compounds and polysaccharides, and thus may have similar perireceptor activities. Phagocytic Organelles General Features. Ciliates are usually heterotrophic; a common means of nutrient uptake is by food vacuole formation, or phagocytosis, a process that comes under the general category of endocytosis (for a review of phagotrophy in Tetrahymena, cf. Nilsson, 1979). Food vacuoles form a t the “cytostome,”an opening at a fixed location in a specialized region of plasma

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

247

Mernbranelle

Membranelle

Mernbronelle

C ytophorynx O r a l Ribs-

---

0 not differentiated

a bristles

63 longitudinal raws of particles

Fig. 33. A simplified diagram of the oral region of Tetruhyrnena showing the location of ciliary membrane differentiations. Cilia are organized into three membranelles (each contains three rows of cilia, but the lengths of the rows vary). Some cilia in the outermost row of the first membranelle have bristles protruding from the ciliary membrane. The orientation of the bristles changes with respect to the oral

ribs as one moves from cilia located close to the oral ribs back along the membranelle. Longitudinal rows of particles are found on the outer rows of cilia in the second and third membranelles. (Reproduced from Sattler and Staehelin, 1974, with permission of The Rockefeller University Press.)

membrane localized somewhere on the cell surface or at the bottom of a pocket in some cases known as the “buccal cavity,” “oral cavity,” or “peristomal cavity.” Surrounding the cytosome, often within the buccal cavity, are specialized groups of cilia which assist in moving food particles into the forming food vacuole, or in filtering out unwanted material (Allen, 1988). These ciliary groups may be few and simple, or numerous and complex, as in the oral membranellar system of Euplotes (Fig. 9). In Paramecium, the ultrastructure of phagocytosis has been characterized extensively by Allen and coworkers (for a comprehensive description, see Fok and Allen, 1988). The region of surface membranes where food vacuoles develop is the “cytopharynx” (Allen, 1988); here the alveolar and epiplasmic layers are absent, so that the cytopharyngeal membrane forms the only barrier between the cytoplasm and the external world. In Tetrahymena, the organization of the oral cavity and associated membranelles has been subjected t o detailed ultrastructural analysis by high voltage microscopy on serial sections and freeze-fracture electron microscopy (Sattler and Staehelin, 1974, 1979) (Fig. 33). Three membranelles are found within the cavity and one membranelle along the margin. A prominent ribbed wall within the cavity leads down into the cytostomal region, where a projecting lip allows food to enter the forming food vacuole but not to leave it. Other ciliates in which electron microscopy has been used to explore the food vacuole forming region and events of early food vacuole formation include Euplotes (Kloetzel, 1974); Vorticella, Epistylis, and Zootham-

nium (McKanna, 1973); and Blepharisma (Dass et al., 1976). Ciliary Bristles. Filamentous, mucoid extensions are common on the surface of sensory kinocilia of vertebrates and invertebrates (Hudspeth and Jacobs, 1979; Moran et al., 1977), where they may serve an adhesive or receptor function. In T. pyriformis and T. thermophila, cilia located in the outermost row of the first membranelle have 37.5-75.0 nm external bristles, associated with oblique E-face arrays of IMPs which may be membrane attachment sites for the bristles (Sattler and Staehelin, 1974; Hufnagel, unpublished) (Figs. 33,34A). This region is anteriormost and should be the first to come into contact with food. The bristles bind polyanionic ferritin (Hufnagel, unpublished) (Fig. 34B). In the region of these arrays and bristles, there is an electron-dense material inserted between the ciliary membrane and the axonemal outer doublet microtubules (Fig. 34C,D). The bristles appear to penetrate the ciliary membrane and attach to the accessory fiber (Sattler and Staehelin, 1974). The bristles and associated structures are found only in the distal portion of the cilia and cover a distance of up to 2 pm along each of 7-11 cilia. Sattler and Staehelin (1974) propose that the ciliary bristles may sense food and trigger a response of oral cilia and that they may also help direct food particles to the nearby oral ribs which lead to the cytostome. The detection of food could be chemosensory, mechanosensory, or both. Linear IMP Arrays i n Tetrahymena. In T . pyriformis, cilia of the second and third oral membranelle have single or complex longitudinal rows of IMPs, possibly linked t o the microtubular doublets by fibrous

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Fig. 34. Oral ciliary bristles of Tetrahymena thermophilu. A Freeze-fracture image showing oblique IMP arrays (0)and associated bristles (b), projecting into the external medium. X 54,000. B: Thin section of cells treated with polyanionic ferritin before fixation. Note that the external ciliary bristles (b) are stained and that they are arranged in rows with an orientation similar to that of the IMP arrays seen in A. ~ 5 5 , 0 0 0 C: . Longitudinal thin section of a single oral cilium, showing that the external bristles (b) are associated with a rod of dense material that runs parallel t o the microtubular axoneme (a) and external to it. x 110,000. D: In transverse sections through cilia

of the first membranelle, near their tips, the eccentric location of the electron-dense rod (arrowheads) is apparent. x 88,100. (C, D: Reproduced from Sattler and Staehelin, 1974, with permission of The Rockefeller University Press.) Fig. 35. Freeze-fracture of the ribbed wall of Tetruhymna thermophila. Two types of linear particle arrays are visible: a single row of larger particles (arrowhead) found on the flanks of the ribs, and a more complex row of smaller particles, found in the valleys between the ribs. x 55,800.

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

Fig. 36. The parasomal sac in Paramecium multimicronucleatum. A: Thin section showing uptake of horseradish peroxidase into parasoma1 sac (ps), near the base of a cilium (c). x 47,500. (Reproduced from Allen, 1988,with permission of Springer-Verlag.) B: Uptake of ferritin into parasomal sac (arrow). Small dots over cell are artifact. x 50,000. (Courtesy of Westcott and Allen, University of Hawaii.) Fig. 37. Tentacle knob of Discophrya collini, longitudinal thin sections. A Stained with ruthenium red. A single layer of surface coat

bridges (Sattler and Staehelin, 1974; Hufnagel, unpublished) (Fig. 30). Although Sattler and Staehelin (1974) propose a role in modifying ciliary beat for these structures, a chemoreceptive function should also be considered. Similar IMP rows are seen on oral cilia of Glaucoma ferox (Montesano et al., 1981) and a number of other ciliate species (Bardele, 1981). In Tetrahymena, linear arrays of P-face IMPs are also found in the grooves and along the posterior flanks of the ribbed wall (Sattler and Staehelin, 1979; Wunderlich and Speth, 1972; Hufnagel, unpublished) (Fig. 35). Single rows of larger particles, spaced about 33.5 nm apart, run along the flanks of the ribs, where underlying ribbons of four microtubules are seen in thin sections. Linear, irregular groups of smaller IMPs, spaced 13.5 nm apart, lie in the grooves. Other linear arrays and IMP bands lie near the ribbed wall. The ribbed wall may have a role in directing food into the cytostome. Parasomal Sacs and Surface Pores Parasomal sacs are deep plasma membrane invaginations located at pellicular pores, often near the bases

249

material is present. Filaments extending from this layer are not present on the haptocyst docking sites (h). x 25,400.B: Treated with Con A. A uniform layer of Con A covers the knob surface, including haptocyst docking sites. x 28,600.C: Treated with WGA. The knob surface has a patchy appearance (arrowhead) and the tentacle shaft appears heavily labeled beginning at the double arrows. x 30,700. (Reproduced from Sundermann and Paulin, 1985,with permission of Journal of Protozoology.)

of cilia (Figs. 2, 4, 36). They are a common feature of ciliates (Pitelka, 1963). Coated vesicles and pits, known in other cells to take part in receptor-mediated endocytosis (Pearse, 19761, are often associated with the sac membrane, e.g., in Tetrahymena (Allen, 1967; Williams and Luft, 1968) (Fig. 4) and Paramecium (Allen, 1978a) (Fig. 36A). Uptake of ferritin, which is normally anionic and binds to positively charged cell surface molecules, has been reported a t surface pores of Tokophyra infusionum (Rudzinska, 1977). In P. multimicronucleatum, horseradish peroxidase (Allen and Fok, 1980) and cationic ferritin, which binds to negatively charged cell surface molecules (Westcott et al., 1985), are taken up into parasomal sacs and coated vesicles (Allen, 1988; Plattner et al., 1985b) (Fig. 36A,B). In T . pyriformis also, cationic ferritin is taken up into parasomal sacs and associated coated vesicles (Nilsson and Van Deurs, 1983). Tentacles of the Suctoria (Fig. 37) One group of ciliates, the suctoria, lack typical phagocytic structures and feed instead by means often-

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tacles, which contact prey organisms, stun the prey with extrusive haptocysts, and serve as a pipe for ingestion of prey cytoplasm. A recent ultrastructural and cytochemical study of the suctorian Discophrya collini suggests that the surface of the terminal knob of the tentacle has chemoreceptors specialized for prey capture (Sundermann and Paulin, 1985). Unlike the tentacle shaft or cell body, the knob is covered with a thin, dense, ruthenium red-staining layer and projecting filaments (Fig. 37A), and it selectively binds Con A (Fig. 37B) and WGA (Fig. 37C), lectins which bind specifically to mannose and acetylated glucosamine, respectively. Both Con A and WGA inhibited attachment of the prey organism T .pyriformis, whereas Helix agglutinin and peanut agglutinin, lectins specific for different sugars, neither inhibited nor bound to the tentacle knob. This suggests that mannose and acetylated glucosamine residues on the knob surface play a role in chemoreceptive processes related to feeding by tentacles. Other experiments indicate that the prey molecules recognized by tentacle knobs may reside on the cilia of Tetrahymena (Sundermann et al., 1986).

Extrusive Organelles (Figs. 38, 39) Ciliates contain a range of species-specific extrusive organelles usually located near the cell surface. These “extrusomes” (Grell, 1973) are a type of membranelimited vesicle which contains various kinds of protein and acid mucopolysaccharide secretions, organized into crystalline arrays. The contents of extrusomes are abruptly discharged from the cell in response to mechanical, electrical, or chemical stimuli (for a review on these organelles, see Hausmann, 1978). Trichocysts of Paramecium (Fig. 38), mucocysts of Tetrahymena (Fig. 39A), ciliary ampules of Euplotes (Fig. 22A), and pexicysts and toxicysts of Didinium are types of extrusomes. Extrusomes appear to be homologous with coelenterate cnidocysts, whose discharge is regulated by interacting mechanical and chemical stimuli (cf. Thorington and Hessinger, 1988; see also Kass-Simon and Hufnagel, this issue). Extrusomes are usually quite numerous; e.g., P . tetraurelia has more than 1,000 trichocysts (Plattner et al., 1984). Extrusomes may participate in such activities as food selection and uptake (Nilsson, 1972), sexual interactions (Tiedke, 1984), secretion of a lorica (Mulisch et al., 19891, cyst formation (cf. Grimes, 1973; Holt and Chapman, 1971; McArdle et al., 1980), formation of attachment structures (cf. Suchard and Goode, 19821, predation (Hara et al., 1985), and protection against predation (Miyake et al., 1990). In all these activities, chemical stimuli for discharge are likely to be important. For example, Didinium discharges its pexicysts and toxicysts when it bumps against a prey organism such as Paramecium or Tetrahymena, but not when it contacts other ciliate species that do not serve as prey; for this reason, Hara et al. (1985) suggested that molecules on the surface of Paramecium serve as stimuli for toxicyst discharge. The molecules released from extrusomes may become incorporated into the cell coat or cell membrane, where they could act as chemoreceptors themselves or as alerting signals for other ciliate cells.

Fig. 38. Trichocysts of Paramecium. A: Diagram based on a tangential thin section through the cortex of Paramecium, showing the distribution of trichocyst docking sites (tt) in relation to cilia (ci) and cortical ridges. a = alveolus. B Diagram based on a vertical thin section through the cortex, showing positioning of the trichocyst a t its docking site. a = alveolus; ci = cilia; tb = trichocyst body; tt = trichocyst tip. C : Diagram of the distribution of membrane particles in the cortical membranes of Paramecium: The arrangement of particles in the P-face of the plasma membrane, including the fusion rosette (c), and surrounding scattered and ring-shaped particle groupings (a, b). D: Arrangement of bridges between the membranes at the docking site, corresponding to IMPs seen in C. a1 = alveolus; e = trichocyst-alveolar bridges; tc = tubular collar. (Reproduced from Hausmann, 1978, with permission of Academic Press.)

Extrusomes are usually docked a t specific sites on the cell surface (Figs. 38A,B, 39B). In freeze-fracture replicas, the plasma membrane a t these sites has a specialized arrangement of IMPs, usually a ring of 811 IMPs around a central IMP, for which the term fusion rosette was coined (Satir, 1974a,b; Satir et al., 197213; Speth and Wunderlich, 1972) (Figs. 38C, 39B). Eufolliculina has a more complex rosette, with 14-16 peripheral IMPs arranged in 2 concentric rings (Mulisch et al., 1989). In E . crassus, the rosettes associated with cortical ampules lack a central particle (Dallai and Luporini, 1981). The electron microscope was useful in correlating the absence of fusion rosettes with the inability of trichocysts of Paramecium and mucocysts of Tetrahymena to exocytose in secretory

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

Fig. 39. Mucocysts of Tetrahymenn thermophila. A: Ultrathin section. On the left, a mucocyst (md) is discharging its contents. On the right, a portion of an undischarged mucocyst (mu), its contents organized into a crystalline array, is present. g = glycogen particles; m = mitochondrion. x 60,000. (Courtesy of Bohmer and Hufnagel, University of Rhode Island.) B: Fusion rosettes of mucocysts of Tetrahymena, seen in freeze-fracture replicas of the P-face and E-face (inset)of the plasma membrane. x 58,900. Inset: x 73,600.

mutants (Beisson et al., 1976; Orias et al., 1983). Surrounding the fusion rosette in Paramecium are two concentric rings of IMPs which may be membranemembrane attachment sites (Allen and Hausman, 1976; Plattner et al., 1973) (Fig. 38C,D). Vilmart and Plattner (1983)have shown by enzymatic digestion and freeze-fracture studies that rosettes and rings of Paramecium are membrane-integrated proteins.

25 1

Fig. 40. Anterior cilium of a mating-competent (starved) Tetrahymena thermophila, showing numerous small ciliary rosettes scattered along the ciliary shaft. x 88,400. Fig. 41. Membrane arrays of mating Euplotes crassus. A Membranellar cilia by which two mating cells are attached to each other. Note the presence of large patches of IMPs. x 74,000. B Loosely organized rosette-like arrays, arranged in lines parallel to the ciliary axis. x 100,000. (Reproduced from Luporini and Dallai, 1981, with permission of Company of Biologists Ltd.)

Holdfast Structures A variety of different ciliates spend a portion of their life cycle attached to a substrate (Hovasse et al., 19721, usually selected for nutritional reasons. For example, a holdfast apparatus attaches Spirochona to appendages of various crustacea (Fahrni, 1984). Other examples of attachment organelles include the contractile stalk of

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Vorticella and other peritrichs, the lorica of Eufolliculina (Mulisch et al., 1989), and the aerial stalk of the aggregated cells of Sorogena (Blanton and Olive, 1983a,b). It is likely that chemoreception is important in selection of a substrate, the initial adhesion event, and development of the attachment structures.

SELECTED BEHAVIORAL REPERTOIRES Motility Swimming Responses. Jennings (1899, 1906) provided early insight into behavioral responses of ciliates to chemical stimuli when he showed that chemical stimulation of the anterior end of a Paramecium can cause an “avoiding reaction.” The normal power stroke of cilia is anterior to posterior, pushing the cell forward. In the avoiding reaction, the cells swim backward due to a reversal of the effective stroke of the somatic cilia. Because they also turn during this response, repeated avoiding reactions tend to produce negative chemokinesis in the presence of such noxious stimuli as non-optimal ionic strength or acidic pH (Van Houten, 1978, 1979; personal communication). The avoidance response mediated by cilia alone in some ciliates is accompanied by a whole body contractile response in other ciliates, such as Stentor, Zoothamnium, Spirostomum, and Vorticella (Ettienne, 1970; Hawkes and Holbertson, 1974; Moreton and Amos, 1979; Sleigh, 1970; Wood, 1975, 1982). In all ciliates examined, the avoidance response is mediated primarily by voltage-dependent calcium channels which permit calcium to enter the cell and interact with the ciliary axoneme to bring about a change in the direction of ciliary beating. Ciliates also show a positive response to chemical stimulation. For example, paramecia show an increase in forward swimming speed due primarily to an increased rate of ciliary beating and a decrease in frequency of avoidance responses. These responses to attractant chemicals, such as potassium acetate, result in positive chemokinesis (Van Houten, 1978, 1979). The net effect of responses to chemicals is either chemoattraction or chemorepulsion. As in bacteria (Adler, 1975), changes in turning frequency underlie most of the observed swimming responses of ciliates (Van Houten and Preston, 1988; Van Houten et al., 1981); unlike the bacteria, swimming speed also is affected by stimuli. While chemokinetic responses (e.g., changes in swimming speed or avoidance rate) appear to predominate, chemotactic responses (oriented movements) also may occur in some ciliates, in response to growth factors (Hellung-Larson et al., 1986), nutrients, pheromones, or sudden increase in concentration of a noxious substance. The existence of oriented responses raises the possibility that the ciliate cell can distinguish between different concentrations of stimulus molecules at different locations on its surface, implying that it may have some way of comparing different sites of chemoreception. However, other models can also be envisioned to explain oriented movements. Chemical Stimuli. Chemical attractants for ciliates are many and varied and include cyclic AMP

(Schulz et al., 1982; Smith et al., 1987), organic nutrients such as acetate, lactate, folic acid, amino acids, peptides, and proteins (Hellung-Larson et al., 1986; Levandowsky and Hauser, 1978; Preston and Usherwood, 1988b; Preston and Van Houten, 1987a; Van Houten and Preston, 1988), cholinergic ligands (Doughty, 1979; Tseng and Levandowsky, 1983), heat-stable components of bacterial cultures (Antipa et al., 19831, and growth factors such as platelet-derived growth factor (Hellung-Larson et al., 1986; Leick and Hellung-Larson, 1985). Tetrahymena accumulates in response to enkephalins and contains a membrane protein which is an analog of vertebrate opiate or enkephalin binding protein (O’Neill et al., 1988; Zipser et al., 1988). Repulsive responses are elicited by hydrophobic compounds (Ueda and Kobatake, 19771, extremes of salt and pH (Van Houten and Preston, 1988; Van Houten et al., 1981), membrane-active organic compounds such as alcohols (Dryl, 1959,1973; Van Houten and Preston, 19881, bitter substances such as quinine and quinidine (see Garcia and Hankins, 1975, and Schaeffer, 1905, cited therein; Van Houten et al., 1981; see also Doughty, 1988), amines (Levandowsky et al., 1984), and the purine nucleotide GTP (Clark and Nelson, 1990). In Tetrahymena, responses to certain chemicals appear to be strain specific (Hellung-Larson et al., 1986). In P. tetraurelia mutants with reduced sensitivity to chemical stimulants have been isolated (DiNallo et al., 1982). Chemoreception of hydrophobic stimuli has been explored electrophysiologically and through membrane fluidity studies in Tetrahymena (Tanabe et al., 1980). Cells may have evolved specific receptors to detect organic chemicals, whereas inorganic ions may act more directly, without the intervention of specific receptors (Machemer, 1989; Van Houten and Preston, 1988). Mechanoreceptive Responses: Implications for Chemoreception. In ciliates, mechanical stimulation to the anterior end of the cell and some chemical stimuli elicit similar avoidance responses (Van Houten, 1979). Therefore, studies of ultrastructural correlates associated with swimming responses to mechanical stimulation in ciliates may offer some insight into the structural basis of responses to chemical stimuli. Electrophysiological studies on Paramecium show that mechanical stimulation elicits local receptor currents. The cell is depolarized when stimulated at or near its anterior end or hyperpolarized when stimulated near or at its posterior end (Eckert et al., 1972). Local anterior inward receptor currents carried primarily by calcium lead to a more global amplified calcium influx that results in ciliary reversal and the avoidance response. Local posterior outward receptor currents carried by potassium lead to an overall hyperpolarization of the cell, resulting in “fast forward movement of the cell, due to more rapid beating of cilia without reversal (Naitoh and Eckert, 1973). Thus, in Paramecium there are two anatomically separated mechanoreceptor potentials (see reviews by Machemer, 1985, 1988; Preston and Saimi, 1990). Similar observations have been made in Stentor (Wood, 1970,1975, 1982, 19891, Didinium (Pape and Machemer, 19861,

ULTRASTRUCTUREAND CHEMORECEPTION IN CILIATES

Stylonychia (de Peyer and Machemer, 1978a; summarized in Deitmer, 1989), and Euplotes (Naitoh and Eckert, 1969). This suggests that specialized membrane regions exist that contain concentrations of mechanoreceptors of different types associated with ion channels of different types. The anterior end of the cell appears to have a localized abundance of mechanoreceptors associated with inwardly conducting calcium channels, while the posterior end appears to have a localized concentration of mechanoreceptors associated with outwardly conducting potassium channels. In contrast, calcium channels mediating the avoidance reaction itself may have a more uniform global distribution over the cell. Chemical substances which call forth swimming responses in ciliates also elicit receptor potentials. For example, folate anions, which attract paramecia, induce changes in their membrane potential (Van Houten, 1979). Similar changes in membrane potential have been detected with other attractants (Van Houten, 1979, 1980; Preston and Van Houten, 1987a). However, a t least for the attractant-induced hyperpolarization, a strong possibility exists that electrogenic pumps rather than ion channels are responsible for membrane potential changes (Preston and Van Houten, 1987a). While electrophysiological studies on deciliated Paramecium and Stylonychia indicate that mechanoreceptors are associated with the cell body rather than the cilia (de Peyer and Machemer, 1978a,b; Dunlap, 1976, 1977; Machemer and Ogura, 1979; Ogura and Machemer, 1980; Ogura and Takahashi, 1976), similar studies on chemoreceptors indicate that some are on the cilia and some on the cell body. The electrophysiological response of P. tetraurelia to acetic, lactic, or glutamic acids was not abolished by deciliation (Preston and Usherwood, 1988b; Preston and Van Houten, 1987b), indicating that receptors for these compounds are mainly on the cell body, with only about 1%at most on the cilia. However, the possibility that some motility receptors are on the cilia cannot be entirely discounted. In particular, cilia of P. tetraurelia have numerous (5,00O/cilium) high affinity, highly specific binding sites for L-[3Hlglutamic acid, a specific attractant (Preston and Usherwood, 1988a). It remains to be shown definitively whether these binding sites are correlated with attractant activity of glutamate. The presence of binding sites is not alone an indication of the presence of chemoreceptor activity. As with mechanoreceptors, there is evidence that somatic chemoreceptors are not distributed uniformly over the cell surface. Preston and Van Houten (1987b) demonstrated an anterior-posterior gradient of sensitivity of the hyperpolarization response to K,-folate, with the greatest sensitivity within or anterior to the buccal cavity. This is consistent with earlier reports of enhanced sensitivity of the anterior end of Paramecium to chemicals affecting behavior (Alverdes, 1922; Dryl, 1969). Unfortunately, electrophysiological studies do not distinguish between binding sites and other parts of the receptor mechanism. In P. tetraurelia, more than seven different behaviorally significant ion channels have been identified (for

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recent reviews, see Hennessey, 1989b; Machemer, 1988; Ramanathan et al., 1988; Saimi and Kung, 1987). Based on extensive electrophysiological studies of behavioral mutants, Kung and collaborators concluded that the avoiding reaction alone utilizes at least four different ion channels of the plasma membrane: the voltage-dependent calcium channel mentioned earlier, a voltage-dependent potassium channel, a calcium-dependent sodium channel, and a calcium-dependent potassium channel (Hinrichsen et al., 1985; Kung and Saimi, 1982). Return to the normal swimming mode also involves the interaction of these channels, as well as enzymatic mechanisms for sequestering or removing calcium from the cytoplasm (Hinrichsen et al., 1985). Ultrastructural Findings Receptors. Where do the chemoreceptor molecules reside? Based on their extensive analysis of chemical attractants which induce swimming responses in Paramecium, Van Houten and co-workers (1983; Schulz et al., 1984) have attempted to develop a ligand that would identify and localize specific chemoreceptors for folate, ultimately to be applied at the ultrastructural level. Efforts to use fluorescence microscopy to characterize the binding pattern of fluorescein-conjugated folate did not reveal anterior-posterior gradients of staining intensity, as might have been expected from electrophysiological measurements (Van Houten and Preston, 1988; Van Houten et al., 1985). However, nonspecific binding could have masked a specific binding pattern related to chemoreceptor activity of folate. To examine this, fluorescein-folate was used to characterize the binding to the surface of a mutant Paramecium that lacks the response to folate. The mutant cells showed almost no discernible fluorescence above background. While this suggests that chemoreceptorrelated folate binding to the cell is uniformly distributed, another possibility is that the mutation broadly affects all folate binding sites, those that are specific to the chemoresponse as well as those that are not. This is a distinct possibility, since folate binding studies indicate the presence of multiple binding activities and since folate-sepharose affinity chromatography yields 10 proteins that bind folate (Schulz et al., 1987). This study illustrates the difficulties which must be surmounted in using cytological methods to answer functional questions concerning chemoreceptors. Realizing that more specificity would be needed for cytochemical studies, Van Houten and co-workers are now working to develop antibodies that recognize the folate and cAMP receptors of Paramecium (Sasner and Van Houten, 1989; Sasner et al., 1988; Van Houten et al., 1991) and to clone and sequence the genes for receptors to folate, CAMP,and other chemical stimuli of Paramecium (cf. Baez et al., 1988). Expression products of the cloned genes could then be used to develop suitable labeled probes for ultrastructural studies. Antibodies which selectively block cAMP receptor function in whole paramecia (Van Houten et al., 1991) may also prove useful in eventual ultrastructural localization of the cAMP receptors of this ciliate (Schlichtherle et al., 1988; Van Houten, 1989). Cyclic AMP receptors may be easier to localize by electron microscopy since affinity

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chromatography has revealed only one CAMPbinding protein from cell body membranes (Gagnon and Van Houten, personal communication). Calcium channels CILIARY REVERSAL CHANNELS. Where do the ion channels mediating ciliary reversal reside? Electrophysiological experiments on ciliated and deciliated Paramecium by Dunlap (1976, 19771, Ogura and Takahashi (19761, and Machemer and Ogura (1979) indicate that calcium channels for ciliary reversal are in the ciliary membrane. Ehrlich et al. (1984, 1988) isolated calcium channels from ciliary membranes of paramecia, inserted them into planar lipid bilayers, and showed that the isolated channels have properties similar to the voltage-dependent calcium channel. Where in the ciliary membrane are these channels located? Plattner (1975) found calcium deposits in association with ciliary granule plaques of Paramecium (Fig. 15). Others also reported calcium affinity sites near ciliary bases when paramecia are fixed in the presence of high calcium concentrations (Fisher et al., 1976; Tsuchiya, 1976; Tsuchiya and Takahashi, 1976). However, the relationship between ciliary plaques and calcium channels for reversal remains unclear. Plaques do not occur on all cilia capable of reversal. Furthermore, freezefracture studies on cilia of Paramecium mutants in which calcium channel function is affected did not reveal alterations in ciliary plaque structure (Byrne and Byrne, 1978). In Paramecium, the voltage-dependent ciliary reversal channel can be reversibly blocked by an anticalmodulin drug, quaternary W-7 (Ehrlich et al., 1988; Hennessey, 1989a; Hennessey and Kung, 19841,and by neomycin (Gustin and Hennessey, 19881, which act externally. The voltage-dependent calcium channel is also inhibited by an antibody raised against ciliary membranes (Ramanathan et al., 1983). Perhaps these inhibitors will provide ligands suitable for future ultrastructural localization of the voltage-dependent calcium channel. OTHER CALCIUM CHANNELS. Where do the ion channels mediating receptor potentials reside? There is no compelling evidence for chemoreceptor-associated calcium channels in ciliates. If they do exist, ultrastructural efforts to localize them are complicated by the multiple roles of calcium in sensory transduction and behavioral responses. In Stylonychia, e.g., there are a t least three different calcium channels (Deitmer, 1989): one activated by mechanical stimulation and two activated by membrane depolarization. Furthermore, the outward K +-current is calcium-dependent. The mechanoreceptor calcium channel is believed to be localized to the anterior somatic membrane, while the voltagedependent calcium channels for ciliary reversal may reside in the ciliary membranes, one in membranellar cilia and one in somatic cilia. Thus, experiments to detect calcium channels by non-specific methods such as the Oschman-Wall (1973) technique are difficult to interpret in relation to receptor functions. Successful ultrastructural methods to localize specific calcium channels will take advantage of known differences in ion selectivity and ion dependence (Deitmer, 1989), or use labeled specific ligands, such as irreversibly bind-

ing selective inhibitors and specific monoclonal antibodies. A number of mutations in P. tetraurelia and P. caudatum affect one or another of the calcium channels (summarized in Saimi and Kung, 1987). It will be only a matter of time before these can be used to develop antibody probes suitable for localizing calcium channels or associated proteins. Potassium channels. In ciliates there are a t least four different potassium channels: one calcium-dependent mechanoreceptor channel and three to four different voltage-dependent channels have been characterized in Paramecium and Stylonychia (Deitmer, 1989). The potassium mechanoreceptor channel is localized mainly to the posterior end of the cell body plasma membrane. The cilia are thought not to have potassium channels (Deitmer, 1989). There are no ultrastructural studies on the localization of these channels. Sodium channels. A mutant of P. tetraurelia called paranoiac over-responds to high external sodium and has an increased inward sodium current (Saimi and Kung, 1980). There may be an alteration in the IMP frequency and distribution of the ciliary plaques (Byrne and Byrne, 19781, but this needs to be reexamined. Since Paramecium is a large cell and freeze-fracture replicas provide small sample sizes, the differences observed between paranoiac and wild type could be due to sampling errors. Cilia without plaques have been seen in wild-type Paramecium by Plattner (1975) and Dute and Kung (reported in Byrne and Byrne, 1978). Furthermore, in Tetrahymena, ciliary plaques vary in size and IMP number in different regions of the cell (Hufnagel, unpublished); this may be true for Paramecium as well. Membrane calcium ATPase. In Paramecium, the membrane hyerpolarization resulting from the attractant stimulus folate may be due to a calcium extrusion pump, possibly a calcium-ATPase (Preston and Van Houten, 1987a; Wright and Van Houten, 1988, 1990). An enzyme with the required characteristics has been identified in cell body membranes (Wright and Van Houten, 1988, 19901, though not yet ultrastructurally. The only site of a calcium-dependent ATPase in cytochemical studies ofP. tetraurelia was at exocytosis sites (Plattner et al., 1977, 1980). In Tetrahymena, a ciliary membrane calcium-ATPase has been correlated with the ciliary plaques (Baugh et al., 1976). Since several different membrane-associated calcium-ATPases have been identified in Paramecium alone (Andrivon et al., 1983; Travis and Nelson, 1986), highly specific antibody probes will be required to localize those specifically associated with chemoreceptors. Flattened cortical alveoli underlie most portions of the plasma membranes of ciliates. The cortical alveoli of Paramecium actively sequester calcium, with strict dependence on ATP and M 8 + , as shown by electron microscopic cytochemistry (Stelly et al., 1991). Calcium sequestration by alveoli may play a role in chemoreception.

Extrusome Discharge Discharge Responses. Trichocyst discharge in Paramecium has been the most extensively analyzed case of extrusome discharge (for recent analytical reviews,

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

see Adoutte, 1988; Satir, 1989). The response t o stimulation is postulated to have the following components: 1) influx of extracellular Ca2+, which may be via ligand-gated Ca2+ channels in the somatic membrane (Plattner et al., 1984);2) subsequent binding of calcium to calmodulin; 3) a movement of H C from the trichocyst interior to the cytosol, with the resultant pH change leading to an expansion of the trichocyst contents; and 4) fusion of the trichocyst membrane with the plasma membrane. The latter correlates with the dephosphorylation of “parafusin,” a cytosolic phosphoprotein (Satir, 1989). The electron microscope contributed significantly to this model. Chemical Stimuli. The natural inducers of extrusome discharge are unknown (Adoutte, 1988). Chemical substances may be the primary signal or may regulate responses to other stimuli. An example of the latter is found in Didinium, which uses extrusomes (pexicysts and toxicysts) for prey capture; because the pexicysts and toxicysts do not discharge when the didinia collide with inedible ciliates, chemical factors are considered important, perhaps in regulating their discharge in response to mechanical stimuli (Hara et al., 1985). While these chemical stimuli are as yet unidentified, those that reside in the membrane of the food organism Paramecium are heat denaturable and readily extracted by Triton X-100 (Hara et al., 1985). The extrusive organelles of some ciliates can be induced to discharge when stimulated in the presence of calcium by externally applied “secretagogues,” including polycationic compounds such as alcian blue (Tiedke, 1976) and aminodextrans (Plattner et al., 1984, 1985a). To induce efficient discharge of trichocysts in Paramecium while maintaining cell viability with few side effects, aminodextrans are most effective. It may be significant that these are polyionic compounds with regularly spaced reactive groups. Ultrastructural Findings. Plattner et al. (1985b) used electron microscopy to follow membrane changes during trichocyst discharge in P. tetraurelia by cationized ferritin and lop6 M aminoethyldextran. Their finding corroborated earlier work that showed that particles of the fusion rosette disperse centrifugally and do not assemble into a rosette until another trichocyst docks a t the same site. Fusion rosettes bind cationized ferritin, but it remains to be determined whether they also selectively bind aminodextrans, or whether aminodextrans exert their effect by binding elsewhere on the cell surface. External calcium is generally required for trichocyst discharge in Paramecium. Satir and co-workers (Satir, 1989; Satir and Oberg, 1978; Satir et al., 1972b, 1973) presented cogent arguments for the presence of ligandgated calcium channels in the plasma membrane, their role in mediating trichocyst discharge, and their association with fusion rosettes. However, this last idea has been rejected by Plattner and colleagues (1975; Matt et al., 19801, in part because in cytochemical studies fusion rosettes do not appear to be sites of calcium accumulation (Plattner and Fuchs, 1975). Fusion rosettes have, however, been correlated ultrastructurally with the presence of a calcium-ATPase (Plattner et al., 1980), suggesting the presence of a calcium pump

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rather than a channel, although a calcium-gated channel cannot be discounted.

Phagocytosis Phagocytic Responses. Organic molecules originating from food sources can cause attractant swimming responses in some ciliates (see earlier Motility Section). Of interest here are interactions of chemical signals with receptors localized to oral structures or used as a basis for food selection and uptake. Studies of phagotrophic behavior in Tetrahymenu have been reviewed by Nilsson (1979). Verity (1991) recently summarized evidence for use of chemical cues for prey selection by ciliates. The ultrastructural and biochemical properties of food vacuole processing have been studied extensively in Paramecium (for reviews, see Fok and Allen, 1988, 1990). Chemical Stimuli. Food vacuoles take up particulate material in an apparently non-specific manner, but in Tetrahymena this uptake can be induced or enhanced by cations (Nilsson, 1972,1976),proteins, polypeptides, RNA, amino acids, polysaccharides and glucose (Ricketts, 1972), pharmacological compounds such as serotonin, caffeine, and dibutyryl CAMP(Rothstein and Blum, 19741, and animal hormones such as histamine (Csaba and Lantos, 1973, 1975). Catecholamine antagonists are inhibitory (Rothstein and Blum, 1974). In Tetrahymena, both mechanical and chemical stimulation may be necessary for food vacuole formation (Ricketts, 1972). In Paramecium, soluble fluorescent proteins, such as bovine serum albumin labeled with fluorescein isothiocyanate, can be taken up into food vacuoles; however, uptake is enhanced by inert latex beads (Fok and Allen, 1988). Food vacuole induction by proteins, polypeptides, and RNA is concentration-dependent. Glutamate, amino acid mixtures, polysaccharides, and glucose are moderately effective, beta-glycerophosphate is slightly effective, and sodium acetate has no effect. On the other hand, in ciliates of the order Apostomatida, the only inducer of food vacuole formation is the exuvial fluid of their crustacean hosts (Bradbury, 1974). In Paramecium, phagocytosis is stimulated by phorbol ester and forskolin (Wyroba, 1987). A large number of substances inhibit phagocytosis; for the most part they probably do not act via cell surface receptors and are not considered here. However, morphine and opioid peptides inhibit phagocytosis in Tetrahymena through a mechanism which may be receptor-mediated (De Jesus and Renaud, 1989; Salaman et al., 1990); a beta-endorphin-like peptide and an opioid receptor have been found in T. thermophila (LeRoith et al., 1982; O’Neill et al., 1988; Zipser et al., 1988). Ultrastructural Findings. Molecules which enhance or inhibit phagocytosis may act by binding to oral cilia and affecting their activity; alternatively, such molecules may bind to particle surfaces and provide active sites for attachment to the cytostomal or oral cavity membrane. In addition, soluble nutrient molecules may attach to membrane receptors in the oral region and be transported along the membrane surface, into the forming vacuole. In any of these situations, specific binding of molecules to receptors on the

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surfaces of cilia or cell soma would be predicted. Cytological evidence for this, especially at the ultrastructural level, is sparse. A close homology exists between phagocytosis a t the cytostome and receptor-mediated endocytosis via coated vesicles; in fact, in some symbiotic ciliates, such as the apostome Hyalophysa chattoni, pinocytotic vesicles rather than food vacuoles form a t the cytostome (Bradbury, 1973). This raises the strong possibility that receptors in the oral region mediate some nutrient uptake into food vacuoles. In Hyalophysa and other ciliates, the membrane in the cytostomal region has a prominent fibrous coating, suggesting the presence of glycoconjugate chemoreceptors. In Paramecium, the membrane of the cytopharynx, where food vacuoles form, has a thin glycocalyx coat (Allen and Fok, 1983), for which a receptor function seems likely. In studies on LR Gold-embedded and frozen thin sections, a monoclonal antibody tagged with colloidal gold labels only membranes of the cytopharynx, nascent and early phagosomes, and nearby discoidal vesicles (Fok and Allen, 1988; Fok et al., 1986). Although the lectin WGA labels the membrane of the nascent food vacuole and the discoidal vesicles, it labels the cytopharyngeal membrane sparsely, if at all; WGA receptors of the cytopharynx may move into the food vacuole membrane during its formation (Allen et al., 1989; Allen, personal communication). While these observations support the view that certain receptors found in the cytopharyngeal region are incorporated into forming food vacuoles, they do not show whether they serve as receptors for food molecules (Allen et al., 1986; Fok and Allen, 1988). In P. tetraurelia, alcian blue stimulates food vacuole formation, binds to phagosomal membranes, and later interferes with normal membrane events during defecation a t the cytoproct (Kaneshiro and Reuter, 1990). This also suggests that in Paramecium membrane receptors are involved in uptake of some substances. The E-face of the membrane of the cytopharynx has a high density of IMPS (-4,600/pm2) compared with the plasma membrane (-150/pm2) (Allen, 1976; Allen and Staehelin, 1981), providing a n interesting parallel to the situation with putative chemoreceptive cilia, mentioned earlier. In Tetrahymena, the lectin Con A, which binds to glucose and mannose, is rapidly taken up into food vacuoles and also appears to interfere with membrane events at the cytoproct (Scott and Hufnagel, 1983; Hufnagel, unpublished). Con A also binds to a commashaped region of the oral cavity near the cytostome (Frisch and Loyter, 1977; Hufnagel, unpublished). These findings suggest that Con A receptors in the oral cavity constitute part of a nutrient selection or food vacuole assembly mechanism for this ciliate. Further characterization of the “oral comma’ at the ultrastructural level is needed. In Tetrahymena, there is strong evidence that selfgenerated mucus contributes to the concentration and uptake of nutrients (Nilsson, 1979), by serving a s a carrier for chemicals which bind to it. Receptors specific for Tetrahymena mucus may exist in the oral region, a possibility which could be explored with elec-

tron microscopic studies which employ gold particles coated with mucus. In many ciliates, food selection may be a n important component of phagocytosis. For example, in Pseudomicrothorax, cells show contact swimming and adhesion responses to filamentous cyanobacteria which are their food source, but not to glass fibers with similar dimensions unless such fibers are coated with waxy starch, a n artificial food substrate (Peck, 1985). The mediator of food selection in some ciliates may be the circumoral cilia. Some circumoral cilia have specialized structures consistent with a role in food selection. For example, as mentioned earlier, in Tetrahymena certain oral cilia have specialized membrane particle arrays (Fig. 34A), associated with external bristles (Fig. 34A-D). In other ciliates, other parts of the feeding apparatus may mediate food selection. For example, in Pseudomicrothorax, scanning electron microscopy reveals that the buccal region contains a n elaborate “cytostomal complex” consisting of a n oval group of corrugations around a central cytostome. Scanning electron microscopy analysis of the interaction of Pseudomicrothorax with its food, filamentous cyanobacteria, indicates that a selective adhesion precedes ingestion and that this is mediated by the cytostomal complex (Peck, 1985).

Receptor-Mediated Endocytosis Endocytotic Responses. In mutant strains of Tetrahymena in which phagocytosis is blocked, uptake of nutrient molecules may occur without food vacuole formation (Rasmussen, 1973,1974; Rasmussen and Orias, 1975, 1976; for a review see Rasmussen, 1976). Such uptake may take place through receptor-mediated endocytosis via pinocytosis for macromolecules, or carrier-mediated transport for low molecular weight compounds (Nilsson, 1979). Chemical Stimuli. The stimuli for pinocytotic uptake have not been systematically explored in ciliates. Anionic and cationic compounds have been found in parasomal sacs and coated vesicles, but whether they stimulated pinocytotic activity is unknown. The receptor-mediated uptake of more specific compounds has not been explored. Ultrastructural Findings. Nutrient uptake via pinocytosis is probably quite common in ciliates. In parasitic ciliates it may be the primary route of uptake. For example, in Terebrospira, a ciliate that lives in and feeds on the exoskeleton of the shrimp Palaemonetes, numerous coated vesicles, a n indicator of receptor-mediated endocytosis, are found near the advancing face of the cell, which also contains acid phosphatase (Bradbury and Goyal, 1976). Bradbury and Goyal(l976) propose that enzymes produced by Terebrospira create host exoskeleton degradation products that bind to the fibrous cell coat of Terebrospira and induce pinocytosis via the coated vesicles. Non-phagosomal nutrient uptake may be via pinocytosis at the parasomal sacs, where ultrastructural studies indicate that coated vesicle formation may occur (Allen, 1976). Since the presence of coated vesicles indicates the existence of receptor-mediated processes, it is likely that specific receptors mediate at least some

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

nutrient uptake in Tetrahymena. As mentioned earlier, electron-dense labels which bind to polyionic cell surface sites, such as anionic and cationic ferritin, and horseradish peroxidase, are taken up into parasomal sacs. Electron microscopy has not yet been applied to the identification or localization of specific receptor macromolecules mediating uptake via parasomal sacs.

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Kuhlmann, 1986; Schulze Dieckhoff et al., 1987; Weischer et al., 1985). In ciliates whose signal molecules are surface-bound, e.g., Paramecium and Tetrahymena, signaling is mediated by direct contact between cells of complementary mating type. This type of mating behavior has been studied extensively in the flagellated protistan Chlamydomonas (Goodenough et al., 1980, 1985; van Sexual Interactions den Ende et al., 1990). In ciliates, some of these mating Behavioral Responses. Chemoreception clearly signals may reside on cilia. When mating cells first has a central role in mating interactions of ciliates. come together it is their cilia which initially make conChemical cues during mating can lead to a variety of tact. This situation continues for some time, e.g., about responses, including changes in motility, secretion of one-half to three-quarters of an hour in the case of T . agglutinins, cell pair formation, fusion of membranes, thermophila kept at 30°C. During this “co~tirnulation’~ and exchange of haploid pronuclei. What has electron period, signaling occurs between the cells (Bruns and microscopy revealed about the identification and local- Brussard, 1974), so it is reasonable to conclude that ization of signals and receptors used at the earliest cilia present signaling molecules. In Paramecium, isostages of the mating process? lated cilia and ciliary membranes are able to stick to Chemical Signals. Sexual interactions in ciliates cilia of complementary mating type (cf. Cohen and Sieare mediated by chemical signals of two types: those gel, 1963; Hiwatashi, 1981; Kitamura, 1988; Kitamura that are soluble in the medium, known as gamones and Hiwatashi, 1976; Takahashi et al., 1974), whereas (Miyake, 1974) or pheromones (Luporini and Miceli, deciliated cell bodies lack this agglutinating reactivity. 1986), and those that are surface-bound to the sexually Thus, the agglutinins appear to be on the cilia and not reactive cells and require direct contact between ga- the cell soma. Whether this ciliary agglutinin is the metes for their interaction with receptors (discussed in same as the mating signal or a separate substance has Miyake, 1981a,b; Watanabe, 1990). The soluble ga- not been established, however (but cf. Goodenough et mones of Blepharisma have been characterized most al., 1985, for Chlamydomonas). While initially contact extensively (cf. Miyake, 1981b). Gamone 1 (blephar- is between cilia only, later cell bodies make contact and mone) (Miyake and Beyer, 1974) is a slightly basic, fuse, so that chemical signals and receptors mediating species-specific glycoprotein, molecular weight 20 kDa, agglutination and fusion probably reside on both the containing glucosamine and mannose, a low content of cilia and the cell soma. Hufnagel (1987) proposed a t glutamic acid, and no tryptophan. Gamone 2 (ble- least three molecular stages of chemoreceptive interpharismone) (Miyake, 1974) is a low molecular weight actions during mating in Tetrahymena, including early compound, 3-(2’-formylamino-5’-hydroxybenzoyl)lac-signaling, later agglutination, and finally membranetate (Kubota et al., 1973), and is common to all species membrane association via extracellular bridging molof Blepharisma (Miyake, 1981a). Gamone 2 is a che- ecules. A parallel situation probably also exists in moattractant and also elicits gamone 1production and other ciliates, including Paramecium. pair formation by cells of the complementary mating The agglutinins of Paramecium have several propertype. Gamone 1 is not a chemoattractant, but does ties; when isolated cilia are added to cells of compleelicit gamone 2 production and pair formation in the mentary mating type, they induce cell clumping (agcomplementary mating type (Miyake, 1981a,b). glutination), selfing (the formation of pairs between The mating pheromones of some Euplotes species are cells of the same mating type), and subsequent events also soluble (Akada, 1986; Heckmann and Kuhlmann, of conjugation. The selfing-inducing ciliary substances 1986; Luporini and Miceli, 1986). For example, the appear to be proteins intrinsic to the ciliary mempheromones of E . raikoui are the basis of a multiple branes (Kitamura, 1988). They can be isolated as memmating type system and consist of related variant gly- brane vesicles. When these vesicles are added to cells of coproteins which may occur as oligomers (Anderson et complementary mating type, they cause pairing (selfal., 1990). Several of these (Er-1, Er-2, Er-3, and Er-11) ing) but not agglutination. Thus, the agglutinin may have been isolated and were either partly character- not yet have been purified. In various species of Paraized or completely sequenced. These have common mecium, only a certain group of ventral cilia has adheamino-terminal residues (Asp) and other sequence ho- sive properties (Hiwatashi, 1969). Ultrastructural Findings. Studies using lectins mologies (Anderson et al., 1990; Concetti et al., 1986; Miceli et al., 1983; Raffioni et al., 1987,19881,and may that bind to specific sugar residues suggested that the resemble the gamones of two other Euplotes species as receptors and surface-bound signals that participate in well as gamone 1ofB. japonicum (Raffioni et al., 1987). mating interactions in some ciliates are glycoconjuOne, Er-1, has been extensively characterized and may gates. For example, in P. tetraurelia, the fusion zone of be a homodimer with a molecular mass of about 9-10 conjugating cells can be labeled with WGA-FITC but kDa. A high affinity receptor for Er-1 has been identi- not Con A-FITC, and labeling is blocked by GlcNAc fied and characterized by binding studies and cross- and WGA but not NeuAc or neuraminidase treatment, linking experiments and may be a protein of Mr = 38 indicating that binding is to GlcNAc residues in the kDa (Ortenzi et al., 1990). Gamones of E. octocarinatus fusion zone (Pape et al., 1988). Early ciliary interachave also been isolated and characterized, but their tions are hardly affected by WGA, but later cell fusion receptors have not yet been identified (Heckmann and is completely blocked. In contrast to these findings,

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Con A blocks conjugation in P. caudatum (Tsukii and Hiwatashi, 1978). Con A also blocks conjugation in T. thermophila, most likely through its binding to specific receptors localized in the region of ciliary and cell soma contact (Casci and Hufnagel, 1988; Frisch and Loyter, 1977; Hufnagel and Lin, 1988a,b; Ofer et al., 1976; Watanabe et al., 1981; Wolfe et al., 1986). While it has been suggested that certain membrane arrays may represent sites of attachment of Con A in Tetrahymena (Wolfe and Grimes, 19791, the ultrastructural parameters of lectin binding have not been explored systematically in any ciliate. Efforts to detect unique ultrastructural properties of mating ciliates related to their early signaling interactions have given mixed results. For example, Watanabe (1981, 1990) found no changes in cell coats of P. caudatum stained with ruthenium red or in the freezefracture appearance of ciliary or plasma membranes, in mating cells. However, in T. thermophila, Hufnagel (1979, 1984) found small IMP rosettes in the ciliary membrane of mating and mating-competent cells but not in non-competent cells, nor in cilia of the nonmating species T. pyrzformis (Fig. 40). These rosettes consist of five to six particles, each 3-4 nm in diameter, and are found only on cilia close to the anterior end of the cell, where they are distributed randomly along the shaft of the cilium. Hufnagel(1984) suggested that they have a role in early signaling between matingreactive cells. They bear a close similarity in size and appearance t o rosettes associated with cellulose export in plants, suggesting a role in the export of polysaccharide signals or receptors used in mating. In membranes of membranellar cilia in mating Euplotes ready to pair, two new types of IMP assemblies appear (Dallai and Luporini, 1983a; Luporini and Dallai, 1981): patches of up to 50 or more IMPs in a loose irregular cluster (Fig. 41A) and rosette-like arrays of a few IMPs on small membrane protrusions (Fig. 41B). Underneath these protrusions, thin sections revealed electron-dense bodies.

SUMMARY AND DISCUSSION Conclusions The precise cell surface locations of chemoreceptors used in specific behavioral responses of cilioprotists remain unknown. Behavioral, electrophysiological, and light microscopic evidence suggest that some receptors are concentrated on cilia while others are present on the cell soma, but this has not yet been corroborated by electron microscopy. Many ciliates have morphologically distinct cilia which could be structurally specialized for chemoreception but such a function has not been demonstrated for any of these cilia. Electron microscopy has permitted identification of receptor sites for certain types of specific ligands, such as lectins, antibodies, and polyanionic and polycationic molecules, but the relationship of these sites to chemoreceptive processes remains elusive. A plethora of structures composed of IMPs has been identified in the cell soma plasma membrane and ciliary membrane of many different ciliate species, but the relationship of these IMP assemblies t o chemoreception is unknown. The cell coat is expected to contain a t least some of the mole-

cules which bind chemical signals, including those that serve as specific receptors and others which may have perireceptor functions, but the chemical and ultrastructural organization of the ciliate cell coat remains poorly understood and the use of electron microscopy to identify and characterize cell coat signal-binding macromolecules remains to be exploited. The implication of polyanionic and polycationic compounds as potent signals and potential receptors seems to be a common thread running through the ciliate chemoreception story.

Future Directions The further elucidation of the ultrastructural parameters of chemoreception in ciliates, as in all cells, awaits the more complete characterization of the biochemistry and physiology of specific chemoreceptor and transduction molecules. In the near future, purification of membrane and glycocalyx macromolecules and cloning of genes of ciliates will lead to the development of specific probes suitable for identification and characterization of components of the receptor modules of ciliates and for localization of chemoreceptors to specific ciliary or cell soma membrane domains. Other useful probes may include lectins, which bind to specific sugar residues, and certain pharmacological reagents such as inhibitors, to which electron-dense markers such as colloidal gold can be attached. Such probes, in concert with the use of mutant cell lines, will then be used to determine whether chemoreception is correlated with any of the structural components of membranes revealed by freeze-fracture and freeze-etch methods. Receptors related to motility responses may be the first to be identified and localized at the electron microscopic level. The whole area of signal transduction, especially the role of second messengers, which has hardly been discussed here, also remains to be explored with the use of specific probes. The common occurrence of polyionic compounds in chemoreceptive processes may suggest new ways of using electron microscopy to explore chemoreception. The incredible organization revealed by electron microscopy in the cilioprotist cell cortex and plasma membrane suggests a number of interesting questions that stretch current paradigms. For example, 1) do processes occurring during chemoreception and chemoresponse in the cilioprotist show any parallels to the complex patterns of odorant signal integration seen in the separately evolved olfactory reception and processing of vertebrates; 2) is there integration between different types of odorant signal reception or between odorant receptors and receptors of other types of stimuli; and 3) does the highly ordered nature of some membrane particle domains have a special significance in relation to chemoreception? In the future, the electron microscope will no doubt be essential to obtain a complete answer to these questions. ACKNOWLEDGMENTS The author expresses her heartfelt appreciation to the contributors of the many fine photographs assembled for this paper, and also to Paul Johnson for his superb photographic assistance and advice. Also

ULTRASTRUCTURE AND CHEMORECEPTION IN CILIATES

greatly appreciated are the many useful suggestions given by Bert Menco, Judy Van Houten, Richard Allen, G. Kass-Simon, and last but not least, my husband and colleague, Robert Zackroff, who also had to eat many dinners out of cans! Some of the original electron micrographs presented here were produced a t the Marine Biological Laboratory, Woods Hole, while the author was supported by an MBL Steps Towards Independence Fellowship.

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