DEVELOPMENTAL GENETICS 13~151-159(1992)

Immunocytochemical Analysis of Secretion Mutants of Tetrahyrnena Using a Mucocyst-Specific Monoclonal Antibody AARON P. TURKEWITZ AND REGIS B. KELLY Department of Biochemistry and Biophysics and Hormone Research Institute, University of California, San Francisco ABSTRACT Dense-core granules represent an adaptation of specialized secretory cells to facilitate stimulus-regulated release of stored proteins. Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates. In Tetrahymena hermophila, the ability to generate mutants in dense-core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis. We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors. We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild-type cells. The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes. In addition, the assay represents a powerful technique for diagnosis of new mutants. 0 1992 Wiley-Liss, Inc. Key words: Tetrahymena, mutants, secretion, mucocysts, immunofluorescence

INTRODUCTION A mark of cellular differentiation during the ontogeny of multicellular organisms is the modification of common features to serve individual tissue types. An example of this is the expansion of the membrane traffic pathway in specialized secretory cells to create unique organelles. Typified by mammalian neuroendocrine and exocrine dense-core secretory granules, these are vesicles that arise from the trans-Golgi network. Existing in many forms, they include chromaffin granules, Weibel-Palade bodies, a-granules, and zymogen granules [Burgess and Kelly, 19871. Their important shared feature is that they remain stored in the cyto-

0 1992 WILEY-LISS, INC.

plasm until stimulated to fuse with the plasma membrane and discharge their highly concentrated contents. In considering the evolution of such a compartment, we would like to identify the elements that endow the granules with their essential abilities: to select and concentrate a specific set of proteins, and to translate an extracellular stimulus into a fusion signal. In both mammals and fish, it has been suggested that content selection depends on sorting signals present in the proteins themselves [Burgess et al., 1987; Sevarino et al., 19891, and a soluble protein has been identified that may facilitate sorting [Chung et al., 19891. A similar role has been proposed for the chromogranidsecretogranin family of proteins, members of which are widely found in dense-core granules [Fischer-Colbrie et al., 1987; Gerdes et al., 19891. The act of granule fusion itself has received great attention [Almers, 19901, yet only a few of the molecular players are known [Schweizer et al., 19891. As has been demonstrated for other complex biological phenomena, the addition of genetic analysis would help resolve and clarify these elements if mutants in the pathway could be isolated. This approach has been particularly successful in studies of the yeast secretory pathway; unfortunately, dense-core granules do not exist, and therefore cannot be studied in this organism. However, the presence of abundant dense-core granules in the ciliates has made these organisms attractive systems to approach these questions, particularly because they can be approached using both genetic and biochemical techniques [Beisson et al., 1976; Aufderheide, 1978; Lefort-Tran et al., 1981; Orias et al., 1983; Satir et al., 1986; VilmartSeuwen et al., 1986; Kerboeuf and Cohen, 1990; Turkewitz et al., 1991; Gutierrez and Orias, 19921. While the function of these granules is not understood, a great deal is known about their structure and composition [Tokuyasu and Scherbaum, 1965; Hausmann, 1978;

Received for publication October 1,1991; accepted November 5,1991. Address reprint requests to Dr. Aaron P. Turkewitz, Department of Biochemistry and Biophysics, UCSF, San Francisco, CA 94143-0448.

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Peterson et al., 1987; Sperling et al., 1987; Tindall et al., 19891. The contents of Tetrahymena thermophila granules, known as mucocysts, have been described; these consist of a number of proteins of widely varying molecular mass, collectively called mucin, many of which may be proteolytically derived from precursors [Collins and Wilhelm, 1981; Maihle and Satir, 1986al. Proteolytic processing is likely to occur in a n immature granule, and the degree of processing is diagnostic of the state of maturation [Turkewitz et al., 19911. Mature mucocysts are ovoid bodies that dock at specific positions along the plasma membrane, defined by the presence of a rosette of intramembrane particles [Pitelka, 1961; Tokuyasu and Scherbaum, 1965; Allen, 1967; Satir et al., 19731. Several thousand mucocysts in a single cell can be stimulated to fuse simultaneously with the plasma membrane, releasing their contents in a n event that can be both induced and detected by Alcian Blue. On the basis of this visualization, exocytosis mutants have been selected, and preliminary characterization has demonstrated that they represent lesions at various stages of the granule pathway [Orias et al., 1983; Maihle and Satir, 1985, 198613; Turkewitz et al., 19911. Since the core of mature mucocysts consists of a distinct protein lattice, they are easily recognized and their distribution can be assessed by electron microscopy. To further this approach, we would like to be able to localize mucocyst proteins in intermediate compartments, which may not contain recognizable cores, and thereby trace the pathway that leads to the synthesis of this specialized secretory compartment. This in t ur n will help map the sites of action of mutations. In this paper, we demonstrate that the use of a monoclonal antibody (mAb) directed against a mucocyst protein facilitates such localization by immunofluorescence. Results obtained using this technique permit us to characterize the secretion mutants based on the nature of the organelles in which mucin protein accumulates.

monoclonal antibody, kindly provided by Dr. E. Marlo Nelsen (University of Iowa) as a n in vitro cell culture supernatant, resulted from fusion IX, clone 4Dl1, subclone F4, and was generated by immunization with Tetrahymena thermophila pellicles prepared after Nozawa and Thompson [1971]. The control for this antibody was a similar supernatant containing an antibody reactive against the human transferrin receptor.

MATERIALS AND METHODS Cells and Cell Culture Tetrahymena thermophila strain SB210 and the derivative exocytosis-defective strains [Orias et al., 19831 were the gift of Eduardo Orias, University of California, Santa Barbara. Strain SB715 was derived from a backcross of the exocytosis-deficient mutant SB255 to a wild-type strain [E. Orias, personal communication] and displays similar mutant phenotype. Cells were grown in 2% proteose peptone, 0.2% yeast extract (Difco Laboratories, Detroit, MI), 10pg/ml ferric chloride at 30°C with stirring. Cell culture density for exponential phase cells was 1-2.5 x 105/ml.

Pulse-Chase Experiments Logarithmically growing cells were transferred to DMC and starved for 2 h at room temperature, then pelleted and resuspended in DMC at a concentration of 106/ml. Cells were labeled with 0.2 mCi/ml b e l ;Bio[35Slmethionine + cysteine ( t r ~ n s - ~ ~ s - l aICN medicals Inc., Irvine, CA) for 5 min. They were then centrifuged through a n underlayered pad of 3% Ficoll in DMC and resuspended in the same volume of DMC containing 2 mg/ml of cysteine and methionine; l-ml aliquots were withdrawn for immune precipitations in 1% Nonidet P-40, 0.4% sodium deoxycholate, 10 mM Tris pH 7.4, 5mM EDTA with protease inhibitors, using fixed Staphylococcus aureus (Zymed Labs, San Francisco, CA) to precipitate the antigen-antibody complexes. A secondary antibody, rabbit antimouse IgG (Bionetics, Kensington MD), was added to bridge the mAb and the Staphylococcus aureus.

Antibodies The rabbit polyclonal antiserum against p80 has been described [Turkewitz et al., 19911. The 4 D l l

Immunofluorescence The protocol followed is based on one provided by Dr. E. Marlo Nelsen (University of Iowa). Cells were rapidly chilled by swirling in ice water, then pelleted, washed once in cold DMC (prepared as 1OX solution): 0.1 mM Na,HPO,, 0.1 mM NaH,PO,, 0.15 mM CaCI,, 0.2 mM sodium citrate, pH 7.1, to which was added 0.1 mM MgCI, and 0.5 mM CaCl,, and suspended in icecold 0.2% Triton X-100 in 50% ethanol for 10 min. After washing twice in 0.1% bovine serum albumin (BSA) in tris-buffered saline (TBS) (0.9% NaCl in 20 mM Tris pH 8.2), they were suspended in 20% hybridoma supernatant in 1% BSA-TBS. These were incubated for 30 min a t room temperature. Cells were washed twice with 0.1% BSA in TBS, and suspended in 1% rhodamine isothiocyanate-coupled goat antimouse IgG (Fisher Biotech, Orangeburg, NY) in 1% BSA-TBS. After 30 min, cells were again washed and suspended in 90% glycerol, 7.5% PBS, 2.5% DABCO [Johnson et al., 19821. They were viewed with a Nikon Diaphot TMD inverted scope. Stimulation of Exocytosis Cells were stimulated with dibucaine or Alcian Blue 8 GX as previously described [Turkewitz et al., 19911. Mucin was purified from cells stimulated with dibucaine as previously described [Turkewitz et al., 19911.

MORPHOLO G Y OF TETRAHYMENA SECRETION MUTANTS

SDS-PAGE and Western Blots Gels were 10% acrylamide 0.27% bis-acrylamide (Laemmli, 1970). Electrophoresed proteins were transferred to nitrocellulose (Towbin et al., 1979) using a dry-blot apparatus (E & K Equipment, Saratoga, CA) at 1 mA/cm2 for 1 h. The blocking buffer was 5% dry milk, 50 mM Tris pH 7.8, 0.02% NP-40, 2 mM CaC1,. Primary antibody incubations were for 1.5 h at room temperature at the following dilutions: anti-p80,1:500; 4 D l l monoclonal anti-80 kD, 1:lOO. Secondary antibodies (antirabbit or antimouse) were obtained coupled to alkaline phosphatase (Promega) and were used at a dilution of 1:7,500. Visualization was with the Bio-Rad AP Color Development Reagent. RESULTS The Monoclonal Antibody 4D11 Recognizes Mucocysts by Immunofluorescence Criteria Monoclonal antibody-secreting hybridomas, generated by immunization using purified T. thermophila ghosts [Nozawa and Thompson, 19711, were screened by immunofluorescence of whole fixed cells [E.M. Nelsen and J. Frankel, personal communication]. One hybridoma supernatant, 4Dl1, was identified th a t produced a punctate pattern suggesting the known distribution of docked mucocysts in these cells. Occasional fixed cells were surrounded by a fluorescent halo [E. Cole, personal communicationl, resembling the capsules that have been visualized using Alcian Blue [Tiedtke, 19761. We have confirmed these results with T. thermophila wild-type strain, SB210, as shown in Figure 1. Indirect immunofluorescence with this antibody results in a n intense signal th at is localized almost entirely at the cell periphery, as clearly seen when the cells are optically sectioned near their midpoint (Fig. lb ) When the microscope focus is shifted away from the midpoint towards a cell edge, the peripheral fluorescence is resolved into a pattern of dotted lines oriented along the long axes of the cell (Fig. lc). Further evidence that the pattern represents docked mucocysts came when we treated cells with reagents known to induce mucocyst discharge, including dibucaine (Fig. 2) and Alcian Blue (not shown). Under these conditions, we observed the immediate (within 30 s ) disappearance of the observed punctate array. No fluorescence was seen when either the monoclonal or rhodamine isothiocyanate-coupled antibody was omitted (not shown).

The m A b Recognizes a Mucocyst Protein of MW 80 kD To identify the protein target for the mAb produced by hybridoma line 4Dl1, mucin was purified from supernatants of stimulated Tetrahymena, and this material was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and probed in a Western blot using the mAb. As shown in Figure 3A,

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Fig. 1. Wild-type (SB210)2'. therrnophilu, fixed and permeabilized. a. Light. b,c. Indirect immunofluorescence using mAb 4Dll. The image in b, showing peripheral fluorescence, results from optical sectioning near the cell midpoints. In c, the plane of focus has been adjusted to lie near the cell membrane, and the fluorescence is resolved into multiple punctate mucocysts. Bar = 18 pM.

the antibody reacts strongly and uniquely with a band of apparent MW 80 kD that is present in purified mucin. A faint band at the same position was seen in a comparable Western blot using whole cell lysate (not shown). The mAb also immunoprecipitated a species of 80 kD from pulse-labeled wild-type cells whose presence continued to increase until at least 45 min after the pulse labelling (Fig. 3B), a s well as some smaller species.

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a

b

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Fig. 2. a: Wild-type (SB210),stimulated with dibucaine for 30 s and then fixed and permeabilized. As in Fig. 1, b and c show immunofluorescence viewed at central and tangential focal planes. The fluorescence is now dramatically reduced relative to Fig. 1,reflecting the rapid synchronous exocytosis of docked mucocysts. Bar = 18 pM.

Secretion Mutants Display Distinct Immunofluorescence Patterns When Probed With the mAb To better understand stages between mucocyst formation and fusion represented by the mucin-secretion mutants, we analyzed them by immunofluorescence using the mAb 4 D l l . Our present survey reinforces some earlier findings, and suggests classes into which the mutants may be grouped on the basis of the pattern of mucin accumulation. SB281 cells, which have been shown to lack recog-

nizable mucocysts [Maihle and Satir, 19851, show far less immunofluorescence than does wild type, and the pattern observed was dramatically different (Fig. 4). Rather than bright peripheral punctate fluorescence, one sees a small number of large faintly fluorescent cytoplasmic bodies. Cells observed in stationary phase contain a larger number of fluorescent cytoplasmic bodies (not shown). The faintness of the signal does not appear to be due to a failure of the SB281 cells to produce the 80-kD protein. Using the mAb for immunoprecipitation from pulse-labeled SB281 cells, we detected synthesis of the 80-kD protein with a delay in appearance similar to that seen in wild-type cells (Fig. 3B). A survey of the additional mutants revealed three more discernible classes, all of which are marked by bright immunofluorescence. A first class, illustrated here by SB285 (Fig. 5 ) , is characterized by heterogeneous cytoplasmic punctate fluorescence, with little or no concentration of fluorescence at the cell surface. Consistent with this, tangential sections show no fluorescent arrays at the periphery. The cytoplasmic bodies appear smaller than the weakly labeled compartment in SB281, but many are larger than the punctate mucocysts seen in wild-type cells. A second class that can be distinguished is illustrated by SB715 (Fig. 6). The bright fluorescence is seen both diffused throughout the cytoplasm and in large heterogeneous vesicles. In addition, these cells show marked peripheral fluorescence, which can be resolved as a n array resembling that seen in wild-type cells. We have isolated, by Percoll gradient centrifugation, SB715 mucocysts that contain a condensed protein matrix like that of wild-type mucocysts; high-magnification images of thin sections of these cells show mucocysts that appear to be docked in positions characteristic of wild-type cells, as well as numerous cytoplasmic mucocysts (not shown). Nonetheless, the wildtype features do not appear to represent a reversion from the initial phenotype, since the cells do not secrete mucin in response to either dibucaine or Alcian Blue (not shown). We have noted that the extensive arrays of docked mucocysts in SB715 are most evident in late exponential phase or stationary cultures, while cytoplasmic fluorescence is seen at all stages. A third class of secretion mutant, illustrated by SB282 in Figure 7, cannot be distinguished from wildtype cells on the basis of their pattern of exclusively peripheral punctate fluorescence, suggesting that mucocysts are synthesized and docked in a normal fashion. Nonetheless, SB282 cells do not release their mucocyst contents upon stimulation and accumulate substantial levels of a previously established mucin precursor in addition to the processed form (not shown).

DISCUSSION A monoclonal antibody, 4Dl1, generated against Tetrahymena ghosts, has been shown to recognize a

MORPHOLOGY OF TETRAHYMENA SECRETION MUTANTS

A

B

Western blot

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Immunoprecipitation with m Ab

SB210

SB281

cntl. anti- 4 D l l p80

mAb

+l

16kD

+ 97kD

+ G8kD

80kD

+

4-97 +68

+ 43kD

*43

+ 29kD

4 2 9

Fig. 3. A. Western blot of purified mucin (5 kg applied per lane.) After transfer, nitrocellulose strips corresponding to single lanes were blotted with control mAb, anti-p80 serum, or 4 D l l mAb and visualized using secondary antibodies coupled to alkaline phosphatase, as described under Materials and Methods. Molecular-weight standards are indicated. B. Immunoprecipitation using mAb 4 D l l from pulselabelled SB210 (wild-type) or SB281 cells. The zero time point corre-

sponds to the end of a 5-minute labelling with [35S]-translabel, as described under Materials and Methods. The control sample shown was taken at the 45-min time point for SB210 cells, using a mAb specific for the human transferrin receptor. The 80-kD species is indicated on the left; molecular weight standards are indicated on the right.

The antibody works well in a n indirect immunofluprotein t h a t is present in mucocysts and released upon stimulation. The mAb recognizes a protein of 80 kD on orescence assay on permeabilized fixed cells, and in Western blots. This band co-migrates with a n abun- wild-type cells it illuminates a pattern of peripheral dant mucin protein that we have called p80 and mucocysts docked along the primary and secondary against which we have raised a polyclonal serum meridians of the cell, similar to that produced using a [Turkewitz et al., 19911. It is likely that the mAb rec- polyclonal serum against a mucocyst protein, p34 ognizes p80, but until this is rigorously demonstrated [Maihle and Satir, 1986bl. Docked mucocysts are we refer to the protein bearing the 4 D l l mAb epitope known to be more widely spaced on primary meridians, as the 80-kD species. In pulse-labeling experiments, a where their docking positions intersperse with the cilnumber of smaller species appear to be specifically im- iary basal bodies, than on the alternating secondary munoprecipitated by mAb 4 D l l in addition to the 80- meridians [Allen, 19671; this feature is also visible in kD protein; these may be cleavage products or may be the immunofluorescence pattern. This pattern changes associated species in a protein complex. The lag in ap- dramatically after cells are stimulated to exocytose, pearance of the 80-kD protein in pulse-labelling exper- and the technique thus offers a sensitive visual assay iments suggests that the protein may derive from a for mucin release. More immediately, the technique permits us to visuprecursor, as has been shown for other Tetrahymena mucin proteins [Collins and Wilhelm, 1981; Turkewitz alize compartments, containing mucin protein, that et al., 19911, a s well as for granule proteins in Parame- may not be evident by microscopic methods which rely cium [Adoutte et al., 19841 and mammals [Orci et al., upon the presence of a recognizable protein core. Similarly, in Paramecium, a monoclonal antibody has been 19871.

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a

a

b

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Fig. 4. SB281 cells, fixed and permeabilized. a. Light. b,c. Indirect immunofluorescence using mAb 4Dl1,as in Fig. 1. Fluorescence is faint and limited to large cytoplasmic bodies. Bar = 18pM.

used to visualize early stages of granule formation by immunoelectron microscopy [Hausmann et al., 19881. In wild-type Tetrahymena, the absence of significant cytoplasmic fluorescence suggests that little or no 80kD protein is present, except in docked mucocysts. Our previous work with another mucocyst protein, p40, however, has suggested that a pool of unprocessed precursor protein is maintained, presumably in a n uncondensed form [Turkewitz et al., 19911.The apparent contradiction would be explained if the 80-kD protein has

Fig. 5. SB285 cells, fixed and permeabilized. a. light. b,c. Indirect immunofluorescence using mAb 4Dl1,as in Fig. 1.The 80kD protein appears to accumulate in numerous heterogeneous cytoplasmic vesicles, and the small number of fluorescent bodies near the plasma membrane do not appear to be ordered in any recognizable array. Bar = 18 pM.

a precursor that is not recognized by the monoclonal antibody. Alternatively, the regulation of synthesis may occur at different steps for different mucin proteins. A third, trivial, explanation might be that protein in the reservoir pool is too dilute to be detected by immunof luorescence. Immunofluorescence imaging of the 80-kD protein in a set of secretory mutants suggests that the mutants

MORPHOLOGY OF TETR4HYMENA SECRETION MUTANTS

a

a

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Fig. 6. SB715 cells, fixed and permeabilized. a. Light. b,c. Indirect immunofluorescence using mAb 4Dl1,a s in Fig. 1. Bright cytoplasmic fluorescence is diffuse as well as contained in heterogeneous vesicles, some of which have the punctate appearance of mucocysts. At the cell membrane, a well-ordered array of mucocysts is seen in tangential section. Bar = 18 pM.

accumulate distinctive mucin-containing vesicles, which may represent intermediates in mucocyst formation. Our work on these mutants has shown that they all display incomplete processing of a mucin precursor; this along with equilibrium density analysis of subcellular membrane fractions has suggested that the mutant cells may accumulate such intermediates. We cannot rule out the possibility that some apparent bio-

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Fig. 7. SB282 cells, fixed and permeabilized. a. Light. b,c. Indirect immunofluorescence using mAb 4Dl1,as in Fig. 1. Fluorescence is largely limited to the periphery in a docked mucocyst array. The pattern is similar to that of wild-type cells. Bar = 18 p M .

synthetic intermediates are instead degradative organelles produced by autophagocytosis. This issue may be clarified in the future by comparing the morphological distribution of 80-kD protein with that of endocytosed markers, and the distribution in subcellular fractions of the 80-kD protein with respect to lysosome markers [Hunseler et al., 19881. Notwithstanding this ambiguity, the clear differences in 80-kD localization within the set of mutants suggest that they fall into recognizable classes which

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reflect the sites of the biosynthetic lesions. One class, represented by the granule-minus mutant SB281 [Maihle and Satir, 19851 has a dramatically reduced level of immunofluorescence, although we could readily demonstrate synthesis of the 80-kD protein. A second mutant, SB283, displays a similar phenotype by immunof luorescence but does not appear to synthesize p80; it does, however, contain cytoplasmic vesicles with dense cores (not shown). The detectable immunofluorescence in SB281 is present in a small number of large cytoplasmic bodies. The low level of this antigen a s assayed by immunof luorescence suggests that, rather than being stored, it is rapidly degraded or secreted. The mutant may not be able to recognize or concentrate proteins which would normally be diverted from a pathway of rapid constitutive release. The absence of another mucocyst protein, p34, has also been noted [Maihle and Satir, 1986b], and we have found that SB281 can rapidly secrete a m u c h precursor [Turkewitz et al., 19911. Two other classes of mutants both show bright cytoplasmic fluorescence in heterogeneous bodies. Some of these may be pre-docked mucocysts, while others appear larger. The classes differ in that one, represented by SB715, also displays peripheral fluorescence in the distinct pattern of docked mucocysts, which have been confirmed by electron microscopy. SB715 contains a population of mucocysts with a buoyant density characteristic of wild-type cells, as well as a population of lower density mucin-containing vesicles [Turkewitz et al., 19911. SB715 can be clearly distinguished from the class represented by SB285 that shows largely cytoplasmic labelling. We hypothesize that the defect in this class acts after that of SB281 but before that of SB715. A fourth class, representing the latest-acting mutations, is not distinguishable from wild type on the basis of mucocyst appearance. The primary defect in this group may be in a signal transduction or fusion apparatus. We note, however, that mutants in this group also appear to accumulate a n elevated level of precursors relative to mature mucin proteins, extrapolating from the single protein we have studied, p40 [Turkewitz et al., 19911. This suggests that incomplete processing is compatible with docking but is nonetheless incompatible with fusion or content release in these mutants.

ACKNOWLEDGMENTS This paper is dedicated to Professor David L. Nanney, on the occasion of his retirement. We are indebted to E. Marlo Nelsen (University of Iowa), who generated, identified, and provided the mAb 4 D l l ; Joseph Frankel (University of Iowa), for making us aware of this antibody; and Eduardo Orias (UC Santa Barbara), who provided the exocytosis mutants. We would also like to thank Luisa Madeddu, Eric Cole,

Adam Linstedt, Linda Matsuuchi and Monica Sauer for valuable help and discussion. APT was supported by a Helen Hay Whitney postdoctoral fellowship. RBK was supported by NIH grants DK33937, NS15927, and NS09878.

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Immunocytochemical analysis of secretion mutants of Tetrahymena using a mucocyst-specific monoclonal antibody.

Dense-core granules represent an adaptation of specialized secretory cells to facilitate stimulus-regulated release of stored proteins. Such granules ...
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