JOURNAL OF MORPHOLOGY 2 1 3 3 - 3 1 (1992)
Video-Enhanced Microscopy of Organelle Movement in an Intact Epit helium KARL J . KARNAKY, JR., LEON T. GARRETSON, AND ROGER G. O'NEIL Department of Anatomy and Cell Biology and the Marine Biomedical and Environmental Sciences Program. Medical University of South Carolina. Charleston, South Carolina 29425 (K.J.K.), and Department of Physiology and Cell Biology, University of Texas Medical School, Houston, Texas 77225 (L.T.G., R.G.O.)
ABSTRACT Digitally enhanced video microscopy has provided improved optical resolution in the study of intracellular organelle/particle movement, particularly in extruded axoplasm and certain thin single cell systems. We report here, for the first time, particle movement in an intact, isolated epithelium, the killifish proximal convoluted tubule. Cytoplasmic particles exhibited predominately unidirectional linear movement approaching several microns in length, sometimes with multiple turns. The velocities of 34 particles measured in 11 cells averaged 0.29 pm/sec (range, 0.007-3.1 pm/sec). Microtubulesthe well-established basis for organelle movement in cells-were present but were sparsely represented in electron micrographs of these cells. Videoenhanced microscopic techniques can now be applied to the study of organelle/ particle movement in a n intact epithelium. o 1992 Wiley-Liss, Inc. Digitally enhanced video imaging techniques combined with differential interference contrast (DIC) microscopy have expanded our understanding of the subcellular particle movements that are fundamental components of many dynamic cellular processes. The most successful applications of these techniques have been in studies either in which the cytoplasm has been extruded from the cells t o make a very thin preparation (e.g., squid giant axon as investigated by Schnapp et al., '85; Hayden and Allen, '84; Hayden et al., '83) or in which the cells under investigation are themselves very thin (e.g., keratocytes as studied by Hayden and Allen, '84; Hayden et al., '83). It is clear from these studies that microtubules play important roles in vesicle movement in eukaryotic cells (Schliwa, '84). The specific absorptive and secretory functions of highly specialized epithelial cells are achieved by dynamically regulating vectorial movement of cytoplasmic vesicles, often involving insertion/retrieval cycles (see Brown, '89; Farquhar, '85; Simons and Fuller, '85). However, application of video imaging tech-
o 1992 WILEY-LISS, INC.
niques to study cytoplasmic organelle movement in intact, isolated epithelia has rarely been attempted. In a pioneering video imaging study, done without digital enhancement, Dibona ('78) described organelle movement in the amphibian urinary bladder. More recently, several laboratories have utilized digital video approaches to estimate the volume of intact, isolated epithelial cells (DiBona et al., '85; Spring, '85; Welling et al., '83). In the present study, we have exploited DIC optics and the Allen Video-Enhanced Contrast System (AVEC-DIC) to examine particle movement in an intact, isolated epithelium, the proximal convoluted tubule of the killifish, Fundulus heteroclitus. When carefully prepared, in vitro tubule preparations permitted the observation and analysis of dynamic organelle movement. Numerous cytoplasmic particle movements were observed with velocities in the range of those Address reprint requests t o Dr. Karl J. Karnaky, Jr., Department of Anatomy and Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2204.
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Electron microscopy Kidney tissues were dissected as above, fixed for 90 min with half-strength Karnovsky’s fixative in 0.1 M Na cacodylate buffer, pH 7.4 (Karnovsky, ’65),washed overnight in buffer, postfixed in 1%osmium tetroxide reduced with potassium ferrocyanide (Karnovsky, ’71), dehydrated, and embedded in Spurr’s low viscosity resin (Spurr, MATERIALS AND METHODS ’69). Thin sections were stained with uranyl Tissue preparation and solutions acetate and lead citrate, and examined with a Specimens of the teleost Fundulus hetero- JEOL 100-CX electron microscope at 60 kV clitus were obtained from Woods Hole Biolog- (Karnaky et al., ’76). ical Laboratory and adapted to 100% artifiAdditional kidney tissues were processed cial sea water for several days as described using the extended osmium tetroxide impregbefore (Karnaky et al., ’76). Forty specimens nation technique of Thiery (Thiery, ’79; Bergwere used for this study. After a n animal was eron and Thiery, ’81) to reveal the ER hetpithed, a kidney was removed and placed in work. Briefly, tissues were fixed by immersion oxygenated Ringer’s solution at room temper- in a 2.5% glutaraldehyde solution containing ature. The Ringer’s solution was a modified tri-sodium citrate and calcium chloride. AfForster’s medium (Forster, ’48) containing ter a 20-min fixation, the kidney fragments (in mM): 135 NaCI, 2.5 KC1, 1.5 CaC12, 1.0 were washed, postfixed for 2 h r a t 4°C in a MgC12, 16.0 NaHC03, 20.0 Trizma base. The buffered 1%osmium tetroxide solution, and pH was 8.25 when gassed with 100% 0 2 . then exposed to 1%osmium tetroxide in disSeveral tubules were dissected with fine tilled water for 5 days at 37°C. Subsequently forceps and transferred to a glass slide, and a tissues were washed, dehydrated, and embeddrop of Ringer’s was added. A #O coverslip ded in Spurr’s medium. Semi-thin sections (thickness = 100 pm) was sealed onto the were examined unstained or stained with slide with a mixture (1:l:l) of Vaseline, lano- lead and uranyl salts in a Hitachi H U - l l C lin, and paraffin (VALAP). In some studies a electron microscope operated a t 60 kV. microperfusion chamber was fabricated by using #O coverslip glass strips as spacers on RESULTS each side of the tissue before covering with Proximal tubule morphology the full coverslip and sealing. Kimwipe Detailed descriptions of the morphology of “wicks” were placed a t opposite ends of this chamber to achieve a constant delivery of the kidney of Fundulus heteroclitus and Fundulus grandis have been published in only a oxygenated Ringer’s to the tissue. few studies (Edwards and Schnitter, ’33; RitVideo-enhanced microscopy ter, ’75; Scully, ’86). According to Edwards The tubule preparations were examined and Schnitter (’331,the renal unit of the with AVEC-DIC microscopy using a n in- kidney consists of a relatively large, wellverted Zeiss Axiomat equipped with a 110 x i vascularized glomerulus, a short neck seg1.3 N.A. planapochromatic objective lens ment, a proximal convolution that comprises (Allen et al., ’81). Real time analogue video nine-tenths of the nephron, and a short duct enhancement and digital image processing portion which leads to a duct proper. We (including mottle substraction, gray scale focused our studies on the readily identifiable s t r e t c h , a n d edge enhancement) were proximal tubule. achieved with a Hamamatsu c1966 Photonic Cells of the proximal tubule are columnar, Microscope System (Allen et al., ’85). The with a centrally located nucleus (Fig. 1). images were recorded simultaneously on both Brush borders, cilia, and junctional coma real time and time lapse video cassette plexes are present. Although microtubules recorder and subsequently photographed with were not abundant, all areas of the cytoplasm a 35-mm camera directly from the video are replete with linear and branching elescreen, using the stop-frame playback mode. ments of smooth endoplasmic reticulum (ER), Particle velocities were measured directly which may represent the “paramembranous from the monitor, using the distances trav- cisternal system” as described by Ericsson elled and the elapse times overlaid by the (’64). The basal cytoplasm is filled with rodcomputer. like mitochondria in close apposition to basal
reported in a number of animal cells. In combination with conventional light and electron microscopic techniques, digitally enhanced imaging techniques should lead to a new understanding of epithelial transport processes. Preliminary versions of this work have been presented in abstract form (Karnaky et al., ’87a,b; ’88).
VIDEO MICROSCOPY OF ORGANELLE MOVEMENT
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Fig. 1. Electron micrograph of proximal convoluted tubule. Centrally located nucleus (N) lies adjacent to the numerous basolateral infoldings (arrows) and associated mitochondria. Brush border (B),cilia (C), and tight junc-
tions (arrowheads) are evident. Supranuclear and basal cytoplasm are replete with organelles, vacuoles, and vesicles. Darkly stained glycogen particles are evident throughout cytoplasm. Bar = 1 pm.
infolds (Fig. 2 and inset). Basal interdigitations are not frequent, a n observation similar to that for proximal tubule cells of Lophius (Ericsson and Olsen, '70). To examine further the extensiveness of the ER membrane system, we used the staining technique of Thiery (Thiery, '79; Bergeron and Thiery, '81). Figure 3 is an electron micrograph from tissue that has been subjected t o long-term exposure to osmium tetroxide. This technique delineates an exten-
sive pattern of electron opacity (endoplasmic reticulum) forming either reticular patterns of long canaliculi or fenestrated saccules. This membrane system appears to connect apical and basal membrane domains of the cell.
Proximal tubule morphology as viewed with AVEC-DIC The best AVEC-DIC microscopy was achieved by examining single tubules in the
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K.J. KARNAKY JR. ET AL
Fig. 2. Higher magnification view of subnuclear region of proximal tubule cell, showing the intimate association of mitochondria, basal infolds, and smooth ER. Basal plasma membranes exhibit numerous infoldings (arrowheads). Smooth ER is more electron-dense than
plasma membrane. Actin filaments (small arrow) and microtubules are distributed sparsely in the basal cytodifferentiation plasm. Bar = 1 IJ-m.Inset: Enlarged ~2.2, between the ER and basal infolds in greater detail.
absence of other overlapping tubules, which degraded the quality of the image. Dense particles, such as pigment granules (Fig. 4c) extruded from the numerous renal pigment cells, also interfered with the image quality and were avoided. We judged the viability of the preparations prior to particle movement analysis by the continuous beating of cilia (which decreased with time) and the incidence of random particle motion within large cytoplasmic vacuoles (which increased with
time). Perfusion with oxygenated Ringer’s could maintain tubules for several hours. Figure 4a shows an AVEC-DIC micrograph in which the optical section is taken at the level of the basal infolds of proximal tubule cells. At the base of each cell there are eight to ten folds, each probably containing groups of two or three mitochondria resolved together. Figure 4b demonstrates that micrographs of high magnification ( 4 , 7 0 0 ~ and ) detail can be achieved with this video tech-
VIDEO MICROSCOPY OF ORGANELLE MOVEMENT
25
Fig. 3. Electron micrograph of proximal convoluted tubule after 5-day osmium tetroxide impregnation in an unstained semithin section (0.3 pm). An extensive electron-opaque membrane system anastomoses throughout the cells, seen both as canaliculi and fenestrated saccules
(most prominent in apical cytoplasm). Two apparent shrunken cells (left) may represent either tubule cells coursing at oblique angles or wandering cells, frequent in this epithelium. B, brush border; L, lumen. Bar = 1 km.
nique: the nucleus, apical cytoplasm, basal infolds, and luminal brush border are clearly revealed. Figure 4c is optically focused on the basal infolds of the proximal tubules cells and shows a minimal “contamination” of pigment cell granules.
nonrandom linear motion were employed for quantitative analysis. The speed of 34 particles measured from 11cells (7 tubules) ranged from 0.007 to 3.1 pmlsec, averaging 0.29 k 0.09 pm/sec. Motion was generally linear with a given particle moving from one point in the cell to another without apparent stopping in between. However, some of the particles moved in a set of steps and even exhibited turning. Figure 5 shows an example of slow particle movement (speed = 0.007 pml sec) with turning. Figure 6 depicts an example of much faster and continuous motion, about 0.16 pmlsec. In rare instances we saw particles moving from one part of the cell to
Particle motion in proximal tubule cells AVEC-DIC images observed from timelapse images replayed from two- to tenfold faster than real time revealed a variety of particle motion, particularly in the apical cytoplasm of these cells. Adhering to the criteria established for viability, only those preparations in which particles exhibited a
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K.J. KARNAKY JR. ET AL.
Fig. 4. AVEC-DIC micrographs of kidney proximal tubule provide optical sectioning capability. a: Optical section of cells bordering lumen. Linear structures perpendicular to long axis of lumen are basal infolds and mitochondria resolved together. Bar = 4 km. b: Enlarged view of apical region shows large vesicles as well as
nuclei, basolateral infolds, and a brush border (arrow). Bar = 2 km. c: At basal pole, extensive aggregates of mitochondria and basal plasma membrane appear as linear elements in parallel. Dark round structures are released pigment granules adhering to outer surface of tubule. B, basal side; L, lumen; N, nucleus. Bar = 2 Km.
VIDEO MICROSCOPY OF ORGANELLE MOVEMENT
27
Fig. 5. Relatively slow particle movement (0.007 pmi sec) is shown in four AVEC-DIC micrographs of a kidney proximal tubule cell photographed directly from the video screen with a 35-mm camera. Particle in motion is enclosed in parentheses and its full excursion is delineated by the black and white line. Time is marked in min:sec:
hundredths of second. The particle moved right to left (toward apical pole of cell) before turning. Total excursion was 7.7 pm, requiring 18.5 min. Time of each frame is as follows: a: 00 hour: 00 minute: 41 seconds: 08 hundredths of a second. b 00:5:32:98. c: 00:12:53:38.d 00:19:09:38. B, basal side; L, lumen. Bar (a) = 2 pm.
another and then returning a t least part way to the point of origin (not shown). Optical sectioning limited our observations to only those events resolved in a given focal plane. -
particles have been detected moving parallel to linear elements where no movement had been detected previously by conventional microscopy in both intact squid giant axon (Allen et al., '82) and in extruded axoplasm preparation (Brady et al., '82). Microtubule-based organelle transport has also been reported in keratocytes (Hayden et al., '83).Further work on dissociated Goplasm from the giant axon led to visualization of bidirectional organelle movements along individual cytoplasmic filaments (Vale et al., '85a; Allen et al., '851, which were subsequently identified as microtubules (Schnapp et al., '85).In many nonep-
DISCUSSION
Intracellular organelle movement in nonepithelial cells Video-enhanced microscopic procedures have been employed to study intracellular organelle movements in living, predominately nonepithelial cells. This emphasis is likely related to the preferred optical qualities innate to thin preparations. Intracellular
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K.J. KARNAKY JR. ET AL
Fig. 6. Relatively fast particle movement (0.16 wm/ sec) is shown in four AWC-DIC micrographs of a kidney proximal tubule cell. Motion and time are labelled as in Figure 5. Right to left motion is more linear than in
Figure 5. Time of each frame is as follows: a: 00 hour: 1 minute: 08 seconds: 58 hundredths of a second. b: 00:1:04:78. c: 00:1:15:38. d 00:1:32:18. B, basal side; L, lumen. Bar = 2 pm.
ithelial cells, organelles can move along microtubules powered by the mechanochemical ATPase kinesin (Vale et al., %a).
intracellular vacuoles. In a second type of movement, the most common form, particles moved from one region of the cell to another with a single linear motion (Fig. 6) or with a series of linear motions (Fig. 5). Occasionally these tandem motions would produce a curved pattern. In a third type of motion, particles moved from one location to another in the cell and then returned toward the point of origin. These particles may have been “tethered” to some linear element in the cytoplasm and were simply recoiling, as suggested for some organelle movements in erythrophores (Stearns, ’88). As shown in Table 1, particle velocities measured in the
Application of video-enhanced microscopic techniques to intact epithelial cells The present study demonstrates that videoenhanced microscopic techniques, such as AVEC-DIC, can be employed to provide extremely detailed views of complex, intact, polarized epithelial cells. With this technique, three basic types of intracellular particle motion were detected. Occasionally, in less than optimal preparations, small particles moved with random motion inside of
VIDEO MICROSCOPY OF ORGANELLE MOVEMENT
TABLE 1. Velocities of organelles or materials in various model systems Cell type
Velocity' (cLm/ sec)
Axon 0.0023-4.6 Mammalian Squid giant 0.3-5.0 (intact) Squid giant O.PZ.2 (dissociated) Connective tissue 0.P3.4 Keratocyte Erythrophore (lysed cell) 0.5-1.0 Epithelium Renal proximal tubule 0.007-3.1 Fibrohlastl 0.1-5.0 epithelial cells2 Epidermoid carcinoma (HeD-2) 0.5-1.0
Reference Williard et al., '74 Allen et al., '82 Vale et al., '85b Hayden et al., '83 Stearns, '88 Present study Bridgman et al., '86 HoDkins et al.. '90
'Velocity of particles was dependent on region of cytoplasm and size of particle. 2Data combined from fibroblasts and epithelial cells grown in culture.
29
bladder, small intestine (Mollgard and Rostgaard, '78), rat renal tubule (Bergeron and Thiery, '81; Bergeron et al., '87), and rat small intestine (Thiery et al., '83). It was implied in these studies that the transport of molecules occurred within the confines of the ER, although no details of mechanisms were offered. Since microtubules and structures of the ER have been shown to be interdependent (Dabora and Sheetz, '88; Terasaki et al., '86) the killifish proximal tubule preparation, with its extensive ER network, should prove to be an excellent model system to examine microtubule/ER interactions. The highly polarized nature of epithelial cells is reflected by the delivery of membrane proteins to separate apical or basolateral cellular domains. In intestinal epithelial cells, microtubules play a key role in the sorting of secretory and membrane proteins and glycoproteins (Bennett et al., '84; Danielsen et al., '83; Eilers et al., '89). While it seems likely that similar microtubule-based organelle motion occurs in killifish renal proximal tubule cells, the elucidation of detailed mechanisms awaits further studies of fluorescently labelled microtubules and/or organelles, as well as studies of the effects of cytoskeletal dsrupting agents on these processes. In a t least some suitable epithelia, video-enhanced microscopy will further our understanding of the basic mechanismb) by which cells achieve vesicular traffic, one of the most fundamental properties of cells.
proximal tubule cells fall within the range reported for organelles or materials in a diverse group of animal cells. Only one previous study has employed video-enhanced microscopy to investigate organelle movement in flattened cultured epithelial cells. This study described movement associated with microtubules, and concluded that organelle transport occurs by a general mechanism found in most cells, with fast axonal transport representing a special case ACKNOWLEDGMENTS (Bridgman et al., '86). The linear particle movement observed in This investigation was supported by an killifish proximal tubule cells implicates a NSF grant (DCB 84091651, an NIH grant cytoplasmic "track" for movement. Linear (DK 40545), a grant from the Cystic Fibrosis cytoskeletal elements were not observed with Foundation, and a grant from Ciba-Geigy, our video techniques, undoubtedly due to the Ardsley, NY. Karl Karnaky (83 226) and compromised optical resolution of such a Roger O'Neil(83 224) were Established Investhick preparation. However, our finding of tigators of the American Heart Association comparatively few microtubules and microfil- during this work. We would like to thank Ms. aments (except in the terminal web and in Terri Westberry, Ms. Torrie Hasher, and Mr. microvilli) in electron micrographs of these Kenneth Orndorff and Ms. J o Ellen Fulkerproximal tubule cells corroborates other mor- son for their excellent technical assistance. phological studies of teleost kidney (Ericsson This research began during Dr. Robert D. and Olsen, '70; Hickman and Trump, '69; Allen's last course in optical methods in biolRitter, ' 75; Scully, '86). ogy a t the Marine Biological Laboratory, We have observed very extensive tubular Woods Hole, Massachusetts. This is contribuand sac-like profiles of ER membranes in tion No. 102 of the Grice Marine Biological these killifish proximal tubule cells. ER net- Laboratory, College of Charleston. works of this extensive nature have been described in a variety of transporting, particLITERATURE CITED ularly absorptive, epithelia using heavy metal Allen, R.D., N.S. Allen, and J.L. Travis (1981) Videoimpregnation techniques. Examples include: enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: A new method capable of frog skin, sheep choroid plexus, rabbit gall-
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analyzing microtubule related motility in the reticulopodial network of Allogromia laticollaris. Cell Motil. lt291-302. Allen. R.D.. J . Metuzals. I. Tasaki. S.T. Bradv. and S.P. Gilbert (1982) Fast Aonal transport in squid giant axon. Science 218t1127-1129. Allen, R.D., D.G. Weiss, J.H. Hayden, D.T. Brown, H. Fujiwake, and M. Simpson (1985) Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: Evidence for an active role of microtubules in cytoplasmic transport. J. Cell Biol. 1OOt1736-1752. Bennett, G., E. Carlet, G. Wild, and S. Parsons (1984) Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins. 111. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucoseinjection. Am J. Anat. 17Ot545-566. Bergeron, M., and G. Thiery (1981) Three-dimensional characteristics of the endoplasmic reticulum of rat renal tubule cells, an electron microscopy study in thick sections. Biol Cell. 42t43-48. Bergeron, M., P. Gaffiero, and G. Thiery (1987) Segmental variations in the organization of the endoplasmic reticulum of the rat nephron. A stereomicroscopic study. Cell Tissue Res. 247t215-225. Brady, S.T., R.J. Lasek, and R.D. Allen (1982) Fast axonal transport in extruded axoplasm from squid giant axon. Science 218: 1129-1131. Bridgman, P.C., B. Kachar, and T.S. Reese (1986) The structure of cytoplasm in directly frozen cultured cells. 11. Cytoplasmic domains associated with organelle movements. J . Cell Biol. 102:1510-1521. Brown, D. (1989) Membrane cycling and epithelial cell function. Am. J. Physiol. 256tFl-Fl2. Dabora, S.L., and M.P. Sheetz (1988) The microtubuledependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell 54t27-35. Danielson, E.M., G.M. Cowell, and S.S. Poulsen (1983) Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules. Biochem. J . 216: 37-42. Dibona, D.R. (1978) Direct visualization of epithelial morphology in the living amphibian urinary bladder. J. Membr. Biol. (Special Issue) 4Ot45-70. Dibona, D.R., K.L. Kirk, and R.D. Johnson (1985) Microscopic investigation of structure and function in living epithelial tissues. Fed Proc. 442693-2703. Edwards, J.G., and C. Schnitter (1933) The renal unit in the hdney ofvertebrates. Am. J. Anat. 53t55-87. Eilers, U., J . Klumperman, and H.-P. Hauri (1989) Nocadazole, a microtubule-activedrug interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2).J . Cell Biol. 108t13-22. Ericsson, J.L.E. (1964) Absorption and decomposition of homologous haemoglobin in renal proximal tubular cells. An experimental light and electron microscopic study. Acta Pathol. Microbiol. Scand. [Suppl.]l68:l121. Ericsson, J.L.E., and S. Olsen (1970) On the fine structure of the aglomerular renal tubule in Lophius piscatorius. Z. Zellforsch. Mikrosk. Anat. 104t240-258. Farquhar, M.G. (1985) Progress in unraveling pathways of Golgi traffic. Annu. Rev. Cell Biol. lt447488. Forster, R.P. (1948) Use of thin kidney slices and isolated renal tubules for direct study of cellular transport kinetics. Science 108:65-67. Hayden, J.H., and R.D. Allen (1984) Detection of single microtubules in living cells: Particle transport can oc-
cur in both directions along the same microtubule. J. Cell Biol. 99r1785-1793. Hayden, J.H., R.D. Allen, and R.D. Goldman (1983)Cytoplasmic transport in keratocytes: Direct visualization of particle translocation along microtubules. Cell Motil. 3:l-19. Hickman, C.P., Jr., and B.F. Trump (1969) The kidney. In W.S. Hoar and D.J. Randall (eds): Fish Physiology. Vol. I. New York: Academic Press, pp. 91-239. Hopkins, C.R., A. Gibson, M. Shipman, and K. Miller (1990) Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum. Nature 346:335-339. Karnaky, K.J., Jr., L.B. Kinter, W.B. Kinter, and C.E. Stirling (1976) Teleost chloride cell. 11. Autoradiographic localization of gill Na,K-ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments. J . Cell Biol. 70.157-177. Karnaky, K.J., Jr., R.D. Allen, L.T. Garretson, and R.G. O'Neil(1987a) Direct visualization of intracellular particleivesicle transport in renal proximal tubule using video-enhanced microscopy. Kidney Int. 31:170. Karnaky, K., R.D. Allen, L. Garretson, and R. O'Neil (198713) Intracellular particle movement in proximal tubule and intestine. Fed. Proc. 46:687. Karnaky, K.J., Jr., R.D. Allen, L.T. Garretson, J.E. Fulkerson, and R.G. O'Neil(1988) Dynamic nature of intracellular particle movement in an isolated epithelium, the killifish proximal tubule. Cell Motil. Cytoskel. 10: 344. Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27:137a. Karnovsky, M.J. (1971) Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. J. Cell Biol. 51: 146a. Mollgard, K., and J. Rostgaard (1978) Morphological aspects of some transporting epithelia suggesting a transcellular pathway via elements of endoplasmic reticulum. J. Membr. Biol. 40t71-89. Ritter, M.B. (1975) Biochemical and morphological changes in the kidney of the mud minnow, Fundulus grandis, during adaptation to fresh water and sea water. Diss. Abstr. Intern. 36t1553B-1554B. Schliwa, M. (1984)Mechanisms of intracellular organelle transport. In J. Shay (ed): Cell and Muscle Motility. Vol. 5. New York: Plenum, pp. 1-82. Schnapp, B.J., R.D. Vale, M.P. Sheetz, and T.S. Reese (1985) Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell 40t455462. Scully, R.R., Jr. (1986) Acomparative study of hydronephrosis and renal compensatory hypertrophy in freshwater and saltwater adapted specimens of the euryhaline teleost, Fundulus heteroclitust An examination of functional demand as a determinant of renal adaptive growth. Diss. Abstr. Int. 46t2167B. Schnapp, B.J., R.D. Vale, M.P. Sheetz, and T.S. Reese (1985) The structure of cytoplasmic filaments in organelle transport in the squid giant axon. Cell 40:455462. Simons, K., and S.D. Fuller (1985) Cell surface polarity in epithelia. Annu. Rev. Cell Biol. 1t243-288. SDrincr. K.R. (1985) The studv of euithelial function bv quantitative light micro"scop;. Pfluegers Arc
Video-Enhanced Microscopy of Organelle Movement in an Intact Epit helium KARL J . KARNAKY, JR., LEON T. GARRETSON, AND ROGER G. O'NEIL Department of Anatomy and Cell Biology and the Marine Biomedical and Environmental Sciences Program. Medical University of South Carolina. Charleston, South Carolina 29425 (K.J.K.), and Department of Physiology and Cell Biology, University of Texas Medical School, Houston, Texas 77225 (L.T.G., R.G.O.)
ABSTRACT Digitally enhanced video microscopy has provided improved optical resolution in the study of intracellular organelle/particle movement, particularly in extruded axoplasm and certain thin single cell systems. We report here, for the first time, particle movement in an intact, isolated epithelium, the killifish proximal convoluted tubule. Cytoplasmic particles exhibited predominately unidirectional linear movement approaching several microns in length, sometimes with multiple turns. The velocities of 34 particles measured in 11 cells averaged 0.29 pm/sec (range, 0.007-3.1 pm/sec). Microtubulesthe well-established basis for organelle movement in cells-were present but were sparsely represented in electron micrographs of these cells. Videoenhanced microscopic techniques can now be applied to the study of organelle/ particle movement in a n intact epithelium. o 1992 Wiley-Liss, Inc. Digitally enhanced video imaging techniques combined with differential interference contrast (DIC) microscopy have expanded our understanding of the subcellular particle movements that are fundamental components of many dynamic cellular processes. The most successful applications of these techniques have been in studies either in which the cytoplasm has been extruded from the cells t o make a very thin preparation (e.g., squid giant axon as investigated by Schnapp et al., '85; Hayden and Allen, '84; Hayden et al., '83) or in which the cells under investigation are themselves very thin (e.g., keratocytes as studied by Hayden and Allen, '84; Hayden et al., '83). It is clear from these studies that microtubules play important roles in vesicle movement in eukaryotic cells (Schliwa, '84). The specific absorptive and secretory functions of highly specialized epithelial cells are achieved by dynamically regulating vectorial movement of cytoplasmic vesicles, often involving insertion/retrieval cycles (see Brown, '89; Farquhar, '85; Simons and Fuller, '85). However, application of video imaging tech-
o 1992 WILEY-LISS, INC.
niques to study cytoplasmic organelle movement in intact, isolated epithelia has rarely been attempted. In a pioneering video imaging study, done without digital enhancement, Dibona ('78) described organelle movement in the amphibian urinary bladder. More recently, several laboratories have utilized digital video approaches to estimate the volume of intact, isolated epithelial cells (DiBona et al., '85; Spring, '85; Welling et al., '83). In the present study, we have exploited DIC optics and the Allen Video-Enhanced Contrast System (AVEC-DIC) to examine particle movement in an intact, isolated epithelium, the proximal convoluted tubule of the killifish, Fundulus heteroclitus. When carefully prepared, in vitro tubule preparations permitted the observation and analysis of dynamic organelle movement. Numerous cytoplasmic particle movements were observed with velocities in the range of those Address reprint requests t o Dr. Karl J. Karnaky, Jr., Department of Anatomy and Cell Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2204.
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K.J. KARNAKY JR. ET AL
Electron microscopy Kidney tissues were dissected as above, fixed for 90 min with half-strength Karnovsky’s fixative in 0.1 M Na cacodylate buffer, pH 7.4 (Karnovsky, ’65),washed overnight in buffer, postfixed in 1%osmium tetroxide reduced with potassium ferrocyanide (Karnovsky, ’71), dehydrated, and embedded in Spurr’s low viscosity resin (Spurr, MATERIALS AND METHODS ’69). Thin sections were stained with uranyl Tissue preparation and solutions acetate and lead citrate, and examined with a Specimens of the teleost Fundulus hetero- JEOL 100-CX electron microscope at 60 kV clitus were obtained from Woods Hole Biolog- (Karnaky et al., ’76). ical Laboratory and adapted to 100% artifiAdditional kidney tissues were processed cial sea water for several days as described using the extended osmium tetroxide impregbefore (Karnaky et al., ’76). Forty specimens nation technique of Thiery (Thiery, ’79; Bergwere used for this study. After a n animal was eron and Thiery, ’81) to reveal the ER hetpithed, a kidney was removed and placed in work. Briefly, tissues were fixed by immersion oxygenated Ringer’s solution at room temper- in a 2.5% glutaraldehyde solution containing ature. The Ringer’s solution was a modified tri-sodium citrate and calcium chloride. AfForster’s medium (Forster, ’48) containing ter a 20-min fixation, the kidney fragments (in mM): 135 NaCI, 2.5 KC1, 1.5 CaC12, 1.0 were washed, postfixed for 2 h r a t 4°C in a MgC12, 16.0 NaHC03, 20.0 Trizma base. The buffered 1%osmium tetroxide solution, and pH was 8.25 when gassed with 100% 0 2 . then exposed to 1%osmium tetroxide in disSeveral tubules were dissected with fine tilled water for 5 days at 37°C. Subsequently forceps and transferred to a glass slide, and a tissues were washed, dehydrated, and embeddrop of Ringer’s was added. A #O coverslip ded in Spurr’s medium. Semi-thin sections (thickness = 100 pm) was sealed onto the were examined unstained or stained with slide with a mixture (1:l:l) of Vaseline, lano- lead and uranyl salts in a Hitachi H U - l l C lin, and paraffin (VALAP). In some studies a electron microscope operated a t 60 kV. microperfusion chamber was fabricated by using #O coverslip glass strips as spacers on RESULTS each side of the tissue before covering with Proximal tubule morphology the full coverslip and sealing. Kimwipe Detailed descriptions of the morphology of “wicks” were placed a t opposite ends of this chamber to achieve a constant delivery of the kidney of Fundulus heteroclitus and Fundulus grandis have been published in only a oxygenated Ringer’s to the tissue. few studies (Edwards and Schnitter, ’33; RitVideo-enhanced microscopy ter, ’75; Scully, ’86). According to Edwards The tubule preparations were examined and Schnitter (’331,the renal unit of the with AVEC-DIC microscopy using a n in- kidney consists of a relatively large, wellverted Zeiss Axiomat equipped with a 110 x i vascularized glomerulus, a short neck seg1.3 N.A. planapochromatic objective lens ment, a proximal convolution that comprises (Allen et al., ’81). Real time analogue video nine-tenths of the nephron, and a short duct enhancement and digital image processing portion which leads to a duct proper. We (including mottle substraction, gray scale focused our studies on the readily identifiable s t r e t c h , a n d edge enhancement) were proximal tubule. achieved with a Hamamatsu c1966 Photonic Cells of the proximal tubule are columnar, Microscope System (Allen et al., ’85). The with a centrally located nucleus (Fig. 1). images were recorded simultaneously on both Brush borders, cilia, and junctional coma real time and time lapse video cassette plexes are present. Although microtubules recorder and subsequently photographed with were not abundant, all areas of the cytoplasm a 35-mm camera directly from the video are replete with linear and branching elescreen, using the stop-frame playback mode. ments of smooth endoplasmic reticulum (ER), Particle velocities were measured directly which may represent the “paramembranous from the monitor, using the distances trav- cisternal system” as described by Ericsson elled and the elapse times overlaid by the (’64). The basal cytoplasm is filled with rodcomputer. like mitochondria in close apposition to basal
reported in a number of animal cells. In combination with conventional light and electron microscopic techniques, digitally enhanced imaging techniques should lead to a new understanding of epithelial transport processes. Preliminary versions of this work have been presented in abstract form (Karnaky et al., ’87a,b; ’88).
VIDEO MICROSCOPY OF ORGANELLE MOVEMENT
23
Fig. 1. Electron micrograph of proximal convoluted tubule. Centrally located nucleus (N) lies adjacent to the numerous basolateral infoldings (arrows) and associated mitochondria. Brush border (B),cilia (C), and tight junc-
tions (arrowheads) are evident. Supranuclear and basal cytoplasm are replete with organelles, vacuoles, and vesicles. Darkly stained glycogen particles are evident throughout cytoplasm. Bar = 1 pm.
infolds (Fig. 2 and inset). Basal interdigitations are not frequent, a n observation similar to that for proximal tubule cells of Lophius (Ericsson and Olsen, '70). To examine further the extensiveness of the ER membrane system, we used the staining technique of Thiery (Thiery, '79; Bergeron and Thiery, '81). Figure 3 is an electron micrograph from tissue that has been subjected t o long-term exposure to osmium tetroxide. This technique delineates an exten-
sive pattern of electron opacity (endoplasmic reticulum) forming either reticular patterns of long canaliculi or fenestrated saccules. This membrane system appears to connect apical and basal membrane domains of the cell.
Proximal tubule morphology as viewed with AVEC-DIC The best AVEC-DIC microscopy was achieved by examining single tubules in the
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K.J. KARNAKY JR. ET AL
Fig. 2. Higher magnification view of subnuclear region of proximal tubule cell, showing the intimate association of mitochondria, basal infolds, and smooth ER. Basal plasma membranes exhibit numerous infoldings (arrowheads). Smooth ER is more electron-dense than
plasma membrane. Actin filaments (small arrow) and microtubules are distributed sparsely in the basal cytodifferentiation plasm. Bar = 1 IJ-m.Inset: Enlarged ~2.2, between the ER and basal infolds in greater detail.
absence of other overlapping tubules, which degraded the quality of the image. Dense particles, such as pigment granules (Fig. 4c) extruded from the numerous renal pigment cells, also interfered with the image quality and were avoided. We judged the viability of the preparations prior to particle movement analysis by the continuous beating of cilia (which decreased with time) and the incidence of random particle motion within large cytoplasmic vacuoles (which increased with
time). Perfusion with oxygenated Ringer’s could maintain tubules for several hours. Figure 4a shows an AVEC-DIC micrograph in which the optical section is taken at the level of the basal infolds of proximal tubule cells. At the base of each cell there are eight to ten folds, each probably containing groups of two or three mitochondria resolved together. Figure 4b demonstrates that micrographs of high magnification ( 4 , 7 0 0 ~ and ) detail can be achieved with this video tech-
VIDEO MICROSCOPY OF ORGANELLE MOVEMENT
25
Fig. 3. Electron micrograph of proximal convoluted tubule after 5-day osmium tetroxide impregnation in an unstained semithin section (0.3 pm). An extensive electron-opaque membrane system anastomoses throughout the cells, seen both as canaliculi and fenestrated saccules
(most prominent in apical cytoplasm). Two apparent shrunken cells (left) may represent either tubule cells coursing at oblique angles or wandering cells, frequent in this epithelium. B, brush border; L, lumen. Bar = 1 km.
nique: the nucleus, apical cytoplasm, basal infolds, and luminal brush border are clearly revealed. Figure 4c is optically focused on the basal infolds of the proximal tubules cells and shows a minimal “contamination” of pigment cell granules.
nonrandom linear motion were employed for quantitative analysis. The speed of 34 particles measured from 11cells (7 tubules) ranged from 0.007 to 3.1 pmlsec, averaging 0.29 k 0.09 pm/sec. Motion was generally linear with a given particle moving from one point in the cell to another without apparent stopping in between. However, some of the particles moved in a set of steps and even exhibited turning. Figure 5 shows an example of slow particle movement (speed = 0.007 pml sec) with turning. Figure 6 depicts an example of much faster and continuous motion, about 0.16 pmlsec. In rare instances we saw particles moving from one part of the cell to
Particle motion in proximal tubule cells AVEC-DIC images observed from timelapse images replayed from two- to tenfold faster than real time revealed a variety of particle motion, particularly in the apical cytoplasm of these cells. Adhering to the criteria established for viability, only those preparations in which particles exhibited a
26
K.J. KARNAKY JR. ET AL.
Fig. 4. AVEC-DIC micrographs of kidney proximal tubule provide optical sectioning capability. a: Optical section of cells bordering lumen. Linear structures perpendicular to long axis of lumen are basal infolds and mitochondria resolved together. Bar = 4 km. b: Enlarged view of apical region shows large vesicles as well as
nuclei, basolateral infolds, and a brush border (arrow). Bar = 2 km. c: At basal pole, extensive aggregates of mitochondria and basal plasma membrane appear as linear elements in parallel. Dark round structures are released pigment granules adhering to outer surface of tubule. B, basal side; L, lumen; N, nucleus. Bar = 2 Km.
VIDEO MICROSCOPY OF ORGANELLE MOVEMENT
27
Fig. 5. Relatively slow particle movement (0.007 pmi sec) is shown in four AVEC-DIC micrographs of a kidney proximal tubule cell photographed directly from the video screen with a 35-mm camera. Particle in motion is enclosed in parentheses and its full excursion is delineated by the black and white line. Time is marked in min:sec:
hundredths of second. The particle moved right to left (toward apical pole of cell) before turning. Total excursion was 7.7 pm, requiring 18.5 min. Time of each frame is as follows: a: 00 hour: 00 minute: 41 seconds: 08 hundredths of a second. b 00:5:32:98. c: 00:12:53:38.d 00:19:09:38. B, basal side; L, lumen. Bar (a) = 2 pm.
another and then returning a t least part way to the point of origin (not shown). Optical sectioning limited our observations to only those events resolved in a given focal plane. -
particles have been detected moving parallel to linear elements where no movement had been detected previously by conventional microscopy in both intact squid giant axon (Allen et al., '82) and in extruded axoplasm preparation (Brady et al., '82). Microtubule-based organelle transport has also been reported in keratocytes (Hayden et al., '83).Further work on dissociated Goplasm from the giant axon led to visualization of bidirectional organelle movements along individual cytoplasmic filaments (Vale et al., '85a; Allen et al., '851, which were subsequently identified as microtubules (Schnapp et al., '85).In many nonep-
DISCUSSION
Intracellular organelle movement in nonepithelial cells Video-enhanced microscopic procedures have been employed to study intracellular organelle movements in living, predominately nonepithelial cells. This emphasis is likely related to the preferred optical qualities innate to thin preparations. Intracellular
28
K.J. KARNAKY JR. ET AL
Fig. 6. Relatively fast particle movement (0.16 wm/ sec) is shown in four AWC-DIC micrographs of a kidney proximal tubule cell. Motion and time are labelled as in Figure 5. Right to left motion is more linear than in
Figure 5. Time of each frame is as follows: a: 00 hour: 1 minute: 08 seconds: 58 hundredths of a second. b: 00:1:04:78. c: 00:1:15:38. d 00:1:32:18. B, basal side; L, lumen. Bar = 2 pm.
ithelial cells, organelles can move along microtubules powered by the mechanochemical ATPase kinesin (Vale et al., %a).
intracellular vacuoles. In a second type of movement, the most common form, particles moved from one region of the cell to another with a single linear motion (Fig. 6) or with a series of linear motions (Fig. 5). Occasionally these tandem motions would produce a curved pattern. In a third type of motion, particles moved from one location to another in the cell and then returned toward the point of origin. These particles may have been “tethered” to some linear element in the cytoplasm and were simply recoiling, as suggested for some organelle movements in erythrophores (Stearns, ’88). As shown in Table 1, particle velocities measured in the
Application of video-enhanced microscopic techniques to intact epithelial cells The present study demonstrates that videoenhanced microscopic techniques, such as AVEC-DIC, can be employed to provide extremely detailed views of complex, intact, polarized epithelial cells. With this technique, three basic types of intracellular particle motion were detected. Occasionally, in less than optimal preparations, small particles moved with random motion inside of
VIDEO MICROSCOPY OF ORGANELLE MOVEMENT
TABLE 1. Velocities of organelles or materials in various model systems Cell type
Velocity' (cLm/ sec)
Axon 0.0023-4.6 Mammalian Squid giant 0.3-5.0 (intact) Squid giant O.PZ.2 (dissociated) Connective tissue 0.P3.4 Keratocyte Erythrophore (lysed cell) 0.5-1.0 Epithelium Renal proximal tubule 0.007-3.1 Fibrohlastl 0.1-5.0 epithelial cells2 Epidermoid carcinoma (HeD-2) 0.5-1.0
Reference Williard et al., '74 Allen et al., '82 Vale et al., '85b Hayden et al., '83 Stearns, '88 Present study Bridgman et al., '86 HoDkins et al.. '90
'Velocity of particles was dependent on region of cytoplasm and size of particle. 2Data combined from fibroblasts and epithelial cells grown in culture.
29
bladder, small intestine (Mollgard and Rostgaard, '78), rat renal tubule (Bergeron and Thiery, '81; Bergeron et al., '87), and rat small intestine (Thiery et al., '83). It was implied in these studies that the transport of molecules occurred within the confines of the ER, although no details of mechanisms were offered. Since microtubules and structures of the ER have been shown to be interdependent (Dabora and Sheetz, '88; Terasaki et al., '86) the killifish proximal tubule preparation, with its extensive ER network, should prove to be an excellent model system to examine microtubule/ER interactions. The highly polarized nature of epithelial cells is reflected by the delivery of membrane proteins to separate apical or basolateral cellular domains. In intestinal epithelial cells, microtubules play a key role in the sorting of secretory and membrane proteins and glycoproteins (Bennett et al., '84; Danielsen et al., '83; Eilers et al., '89). While it seems likely that similar microtubule-based organelle motion occurs in killifish renal proximal tubule cells, the elucidation of detailed mechanisms awaits further studies of fluorescently labelled microtubules and/or organelles, as well as studies of the effects of cytoskeletal dsrupting agents on these processes. In a t least some suitable epithelia, video-enhanced microscopy will further our understanding of the basic mechanismb) by which cells achieve vesicular traffic, one of the most fundamental properties of cells.
proximal tubule cells fall within the range reported for organelles or materials in a diverse group of animal cells. Only one previous study has employed video-enhanced microscopy to investigate organelle movement in flattened cultured epithelial cells. This study described movement associated with microtubules, and concluded that organelle transport occurs by a general mechanism found in most cells, with fast axonal transport representing a special case ACKNOWLEDGMENTS (Bridgman et al., '86). The linear particle movement observed in This investigation was supported by an killifish proximal tubule cells implicates a NSF grant (DCB 84091651, an NIH grant cytoplasmic "track" for movement. Linear (DK 40545), a grant from the Cystic Fibrosis cytoskeletal elements were not observed with Foundation, and a grant from Ciba-Geigy, our video techniques, undoubtedly due to the Ardsley, NY. Karl Karnaky (83 226) and compromised optical resolution of such a Roger O'Neil(83 224) were Established Investhick preparation. However, our finding of tigators of the American Heart Association comparatively few microtubules and microfil- during this work. We would like to thank Ms. aments (except in the terminal web and in Terri Westberry, Ms. Torrie Hasher, and Mr. microvilli) in electron micrographs of these Kenneth Orndorff and Ms. J o Ellen Fulkerproximal tubule cells corroborates other mor- son for their excellent technical assistance. phological studies of teleost kidney (Ericsson This research began during Dr. Robert D. and Olsen, '70; Hickman and Trump, '69; Allen's last course in optical methods in biolRitter, ' 75; Scully, '86). ogy a t the Marine Biological Laboratory, We have observed very extensive tubular Woods Hole, Massachusetts. This is contribuand sac-like profiles of ER membranes in tion No. 102 of the Grice Marine Biological these killifish proximal tubule cells. ER net- Laboratory, College of Charleston. works of this extensive nature have been described in a variety of transporting, particLITERATURE CITED ularly absorptive, epithelia using heavy metal Allen, R.D., N.S. Allen, and J.L. Travis (1981) Videoimpregnation techniques. Examples include: enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: A new method capable of frog skin, sheep choroid plexus, rabbit gall-
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analyzing microtubule related motility in the reticulopodial network of Allogromia laticollaris. Cell Motil. lt291-302. Allen. R.D.. J . Metuzals. I. Tasaki. S.T. Bradv. and S.P. Gilbert (1982) Fast Aonal transport in squid giant axon. Science 218t1127-1129. Allen, R.D., D.G. Weiss, J.H. Hayden, D.T. Brown, H. Fujiwake, and M. Simpson (1985) Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: Evidence for an active role of microtubules in cytoplasmic transport. J. Cell Biol. 1OOt1736-1752. Bennett, G., E. Carlet, G. Wild, and S. Parsons (1984) Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins. 111. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucoseinjection. Am J. Anat. 17Ot545-566. Bergeron, M., and G. Thiery (1981) Three-dimensional characteristics of the endoplasmic reticulum of rat renal tubule cells, an electron microscopy study in thick sections. Biol Cell. 42t43-48. Bergeron, M., P. Gaffiero, and G. Thiery (1987) Segmental variations in the organization of the endoplasmic reticulum of the rat nephron. A stereomicroscopic study. Cell Tissue Res. 247t215-225. Brady, S.T., R.J. Lasek, and R.D. Allen (1982) Fast axonal transport in extruded axoplasm from squid giant axon. Science 218: 1129-1131. Bridgman, P.C., B. Kachar, and T.S. Reese (1986) The structure of cytoplasm in directly frozen cultured cells. 11. Cytoplasmic domains associated with organelle movements. J . Cell Biol. 102:1510-1521. Brown, D. (1989) Membrane cycling and epithelial cell function. Am. J. Physiol. 256tFl-Fl2. Dabora, S.L., and M.P. Sheetz (1988) The microtubuledependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell 54t27-35. Danielson, E.M., G.M. Cowell, and S.S. Poulsen (1983) Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules. Biochem. J . 216: 37-42. Dibona, D.R. (1978) Direct visualization of epithelial morphology in the living amphibian urinary bladder. J. Membr. Biol. (Special Issue) 4Ot45-70. Dibona, D.R., K.L. Kirk, and R.D. Johnson (1985) Microscopic investigation of structure and function in living epithelial tissues. Fed Proc. 442693-2703. Edwards, J.G., and C. Schnitter (1933) The renal unit in the hdney ofvertebrates. Am. J. Anat. 53t55-87. Eilers, U., J . Klumperman, and H.-P. Hauri (1989) Nocadazole, a microtubule-activedrug interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2).J . Cell Biol. 108t13-22. Ericsson, J.L.E. (1964) Absorption and decomposition of homologous haemoglobin in renal proximal tubular cells. An experimental light and electron microscopic study. Acta Pathol. Microbiol. Scand. [Suppl.]l68:l121. Ericsson, J.L.E., and S. Olsen (1970) On the fine structure of the aglomerular renal tubule in Lophius piscatorius. Z. Zellforsch. Mikrosk. Anat. 104t240-258. Farquhar, M.G. (1985) Progress in unraveling pathways of Golgi traffic. Annu. Rev. Cell Biol. lt447488. Forster, R.P. (1948) Use of thin kidney slices and isolated renal tubules for direct study of cellular transport kinetics. Science 108:65-67. Hayden, J.H., and R.D. Allen (1984) Detection of single microtubules in living cells: Particle transport can oc-
cur in both directions along the same microtubule. J. Cell Biol. 99r1785-1793. Hayden, J.H., R.D. Allen, and R.D. Goldman (1983)Cytoplasmic transport in keratocytes: Direct visualization of particle translocation along microtubules. Cell Motil. 3:l-19. Hickman, C.P., Jr., and B.F. Trump (1969) The kidney. In W.S. Hoar and D.J. Randall (eds): Fish Physiology. Vol. I. New York: Academic Press, pp. 91-239. Hopkins, C.R., A. Gibson, M. Shipman, and K. Miller (1990) Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum. Nature 346:335-339. Karnaky, K.J., Jr., L.B. Kinter, W.B. Kinter, and C.E. Stirling (1976) Teleost chloride cell. 11. Autoradiographic localization of gill Na,K-ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments. J . Cell Biol. 70.157-177. Karnaky, K.J., Jr., R.D. Allen, L.T. Garretson, and R.G. O'Neil(1987a) Direct visualization of intracellular particleivesicle transport in renal proximal tubule using video-enhanced microscopy. Kidney Int. 31:170. Karnaky, K., R.D. Allen, L. Garretson, and R. O'Neil (198713) Intracellular particle movement in proximal tubule and intestine. Fed. Proc. 46:687. Karnaky, K.J., Jr., R.D. Allen, L.T. Garretson, J.E. Fulkerson, and R.G. O'Neil(1988) Dynamic nature of intracellular particle movement in an isolated epithelium, the killifish proximal tubule. Cell Motil. Cytoskel. 10: 344. Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27:137a. Karnovsky, M.J. (1971) Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. J. Cell Biol. 51: 146a. Mollgard, K., and J. Rostgaard (1978) Morphological aspects of some transporting epithelia suggesting a transcellular pathway via elements of endoplasmic reticulum. J. Membr. Biol. 40t71-89. Ritter, M.B. (1975) Biochemical and morphological changes in the kidney of the mud minnow, Fundulus grandis, during adaptation to fresh water and sea water. Diss. Abstr. Intern. 36t1553B-1554B. Schliwa, M. (1984)Mechanisms of intracellular organelle transport. In J. Shay (ed): Cell and Muscle Motility. Vol. 5. New York: Plenum, pp. 1-82. Schnapp, B.J., R.D. Vale, M.P. Sheetz, and T.S. Reese (1985) Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell 40t455462. Scully, R.R., Jr. (1986) Acomparative study of hydronephrosis and renal compensatory hypertrophy in freshwater and saltwater adapted specimens of the euryhaline teleost, Fundulus heteroclitust An examination of functional demand as a determinant of renal adaptive growth. Diss. Abstr. Int. 46t2167B. Schnapp, B.J., R.D. Vale, M.P. Sheetz, and T.S. Reese (1985) The structure of cytoplasmic filaments in organelle transport in the squid giant axon. Cell 40:455462. Simons, K., and S.D. Fuller (1985) Cell surface polarity in epithelia. Annu. Rev. Cell Biol. 1t243-288. SDrincr. K.R. (1985) The studv of euithelial function bv quantitative light micro"scop;. Pfluegers Arc
