Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, pp. 4976-4980, December 1975
Cell Biology
Membrane-microtubule interactions: Concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins (cultured ovarian granulosa cells/electron microscopy/vinblastine/fluorescein thiocarbamyl colchicine)
DAVID F. ALBERTINI AND JOHN I. CLARK Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115
Communicated by Don W. Fawcett, September 2, 1975
The relationship between microtubules and concanavalin A surface receptors during concanavalin A cap ping in primary cultures of rabbit ovarian granulosa cells was examined by electron microscopic and fluorescence labeling techniques. Cells treated with concanavalin A and hemocyanin at 40 or 370 and then incubated at 370 for 1 hr formed large juxtanuclear caps that were observed with shadow cast replicas of the cell surface. Thin section analysis of capped cells revealed an abundance of microtubules immediately beneath the cap which were arranged approximately perpendicular to the plane of the membrane. The capping process was unaffected by the antimicrotubule agents colchicine or vinblastine. Further, vinblastine treatment of capped calls resulted in the formation of numerous paracrystals that were confined to the cytoplasm underlying the capped region of the membrane; uncapped cells displayed paracrystals that were randomly dispersed in the cytoplasm. Exposure of fixed cells to fluorescein thiocarbamyl colchicine, which localizes colchicine binding proteins, revealed an intensely fluorescent region that corresponded to the cap; this staining pattern was absent in uncapped cells. These findings indicate that concanavalin A mediated capping modifies the cytoplasmic disposition of microtubules and colchicine binding proteins. Further, it is suggested that the capped region of the plasma membrane is a preferred site of microtubule polymerization. ABSTRACT
of the cytoplasmic microtubule system such that these structures become specifically associated with the capped portion of the plasma membrane. Using a sensitive fluorescent probe for localizing colchicine binding proteins, it was found that tubulin (a colchicine binding protein) is preferentially concentrated in the capped area of the cell. These results suggest that the capped membrane domain may be a preferred site of microtubule polymerization.
MATERIALS AND METHODS Granulosa cells were obtained from preovulatory ovarian follicles (0.9-1.1 mm diameter) of adult nonvirgin Dutch belted rabbits and cultured as described (10). After dissociation of granulosa cells from follicular fluid and eggs by passage through a graded series of micropipettes, cells (from four ovaries) were pooled in fresh medium and cultures seeded by adding four drops, containing approximately 1 X 105 cells, to 18-mm glass coverslips contained in 35 X 10 mm tissue culture dishes. The cells were allowed to attach for 1 hr, after which 2.5 ml of culture medium was added to each dish. The culture medium consisted of McCoy's 5A medium (modified) supplemented with 1% L-glutamine (200 mM stock), 100 units/ml of penicillin, 5 Ag/ml of streptomycin sulfate, and 15% serum obtained from adult female nonvirgin rabbits. Cultures were incubated at 370 in 95% air, 5% CO2; the medium was changed every 2 days. All experiments were performed on 5-day cultures showing predominantly flattened epithelial cell sheets with little cellular overlapping. For thin section and replica preparations, the labeling protocol of Ryan et al. (11) was used. Granulosa cell cultures were washed three times in Earle's Balanced Salt Solution (EBSS) and incubated at 40 or 370 for 1 hr in the presence or absence of colchicine (10-5 M) (Sigma) or vinblastine sulfate (10-5 M) (Eli Lilly Co.). The cells were then exposed to Con A (100 ,.g/ml) (Sigma, grade IV) in EBSS for 10 min at 40 or 370, serially rinsed three times in EBSS, and incubated with either hemocyanin (500 Atg/ml) or horseradish peroxidase (200 ,4g/ml) (Sigma, type VI) in EBSS for 10 min at 40 or 370. After three additional washes with EBSS, the cultures were fixed in 1% glutaraldehyde immediately or after incubation at 370 for 10, 30, or 60 min in EBSS. The inherent distribution of Con A binding sites was determined by labeling cells with Con A and hemocyanin (Con A/H) as above, but after a 10-min fixation in 1% paraformaldehyde in EBSS at 40. The reversibility of Con A binding was evaluated in all experiments by incubating cultures in 0.05 M a-methylD-glucopyranoside (Sigma) for 60 min after the addition of visual markers. In vinblastine and colchicine experiments,
Cytoplasmic microtubules have been implicated in the control of the spatial distribution of membrane transport carriers and lectin receptors in several cell types (1, 2). Treatment of cells with the tetravalent plant lectin concanavalin A (Con A), under appropriate conditions, is known to cause a polar accumulation of receptors which is referred to as a cap. When labeled with low doses of Con A at 370, a variety of cell types, including lymphocytes (3, 4), polymorphonuclear leukocytes from rabbits or beige mice (5), and amebocytes (6), cap spontaneously. Lymphocytes and virally transformed cells are unable to cap in the presence of high doses of Con A at 370. Under these conditions, capping ensues only after treatment with vinca alkaloids, low temperature, or colchicine (3, 7, 8), conditions known to disrupt microtubules (9), suggesting that microtubules participate in the modulation of cell surface receptor mobility. Morphological support for a microtubule-membrane association during capping has been difficult to obtain, largely due to the scarcity of microtubules in the cell types previously studied. We report here that Con A cap formation on ovarian granulosa cells in vitro is accompanied by a reorganization Abbreviations: Con A, concanavalin A; Con A/H concanavalin A by labeling with hemocyanin; EBSS, Earle's Balanced Salt Solution; FITC, fluorescein isothiocyanate; FTCCLC, fluorescein thiocarbamyl colchicine. treatment followed
4976
Proc. Nat. Acad. Sci. USA 72 (1975)
Cell Biology: Albertini and Clark
the drugs were present in all labeling solutions and solutions used after incubation, and in one set of experiments colchicine or vinblastine was added 30 min after incubation and the cultures were fixed 60 min later. For the demonstration of peroxidase activity, cells were reacted with diaminobenzidine (0.5 mg/ml) (Sigma) in 0.1 M Tris buffer (pH 7.4) containing 0.01% H202 for 15 min, washed three times in buffer, and photographed under phase and bright field optics. Phase observations were also made on living cells during the period after incubation. All cultures were post-fixed in 1% aqueous OS04 for 15 min, dehydrated in a cold series of ethanols, and either embedded in an Epon 812-araldite mixture for thin section examination after staining with uranyl acetate and lead, or dried from amyl acetate using a hot air stream to make surface replicas according to the method of Smith and Revel (12). The fluorescein thiocarbamyl colchicine (FTC-CLC) used to localize intracellular colchicine binding proteins was prepared in the following fashion. 1 ml of 10-3 M deacetyl colchicine was dissolved in 95% ethanol and added to 1 ml of an ethanolic solution of 7 X 10-3 M fluorescein isothiocyanate-I (FITC) (Sigma) and was permitted to react at room temperature for 60 min. The FTC-CLC was separated by thin-layer chromatography and eluted from the plates with distilled water to give a final preparation containing 5 X 10-3 M FTC-CLC. This preparation was compared with unlabeled colchicine in a variety of cell types and was found to be identical in all systems tested.* For labeling, cells that had been incubated for 0, 10, 30, and 60 min after treatment with Con A at 370 were fixed with acetone for 5 min at -10° and further incubated for 30 min in 5 X 10-6 M FTC-CLC in 0.14 M phosphate buffer, pH 7.2. In some experiments, cultures were incubated in EBSS for 1 hr, labeled for 10 min in Con A-FITC (10 or 100,ug/ml) (Miles-Yeda) at 370, washed three times with EBSS, incubated for 30 min at 370, and then fixed in acetone for 5 min. All cultures treated with Con A-FITC or FTC-CLC were observed with a Leitz microscope equipped with a Ploem vertical illuminator and fluorescence optics. Photographs were recorded on Kodak tri-X film. The control and experimental treatments using FTC-CLC are depicted in Table 1. RESULTS Observation of living or glutaraldehyde fixed cells 30 min after Con A treatment revealed the presence of numerous surface protrusions localized directly over or adjacent to the nucleus (Fig. 1). Cells labeled with peroxidase for the demonstration of Con A binding sites showed under bright-field optics that this area corresponds to the Con A cap (Fig. 2). Surface replicas of capped cells treated with hemocyanin as a visual marker revealed the presence of a 7- to 12-Am spherical mass of marker molecules generally situated near the cell center (Fig. 3). Close inspection of capped cells showed a complete clearing of hemocyanin molecules from the cell periphery (Fig. 3, inset), with clusters of hemocyanin confined to the cap. a-D-Methyl glucopyranoside-treated cultures showed few hemocyanin molecules on the cell surface, and cultures pretreated with paraformaldehyde prior to Con A labeling displayed a homogeneous distribution of hemocyanin. The capped portion of the granulosa cell surface was highly irregular in contour, and marker molecules were observed bound to coated and uncoated surface invag*
J.
I. Clark and D. Garland, manuscript in preparation.
4977
Table 1. Specificity of FTC-CLC binding to capped and uncapped granulosa cells*
Capped Uncappedt
Controls
FTC-CLC Comp.
0 0
+
+++
+
++
FTC-CLC
Cells were treated with medium lacking FTC-CLC or containing sodium fluorescein alone (Controls). 10-6 M FTC-CLC was competed with 1O-3 M unlabeled colchicine in the same medium (FTC-CLC Comp.). *Fluorescence intensity was arbitrarily scored on a 0 to + + + + basis, where 0 = no apparent fluorescence and ++++ = maximum fluorescence. t Capped cells were treated with Con A (100 gg/ml) for 10 min at 370, washed three times in EBSS, and incubated 30 min at 370 in EBSS before fixation and FTC-CLC treatment as described in the text. Uncapped cells were treated as above except in the absence of Con A.
inations as well as lining more deeply situated tubulovesicular profiles seen in thin sections (Fig. 4). Sections approximately normal to the capped surface revealed an abundance of perpendicularly oriented 24-nm microtubules beneath the capped portion of the plasma membrane. Microtubules were present in untreated granulosa cells but were not as abundant as in Con A-treated cultures and usually were arranged parallel to the long axis of the cell. In granulosa cells actively internalizing Con A caps, hemocyanin-laden vesicles were often seen adjacent to microtubules. In more oblique sections, microtubules and microfilaments (6 and 10 mm in diameter) were observed to course along the cell periphery in close association with the cell membrane (Fig. 5). Cultures capped in the presence of colchicine or vinblastine displayed large, more peripherally located hemocyanin caps that characteristically had smooth contours. Microtubules were rarely observed in drug-treated cultures. The addition of vinblastine to capped cultures (30 min after incubation) resulted in the formation of tubulin paracrystals associated with the capped region of the cell (Fig. 6). The paracrystalline inclusions were comprised of tubular elements having an internal diameter of 20-26 nm, in fair agreement with previously published reports (13). Juxtanuclear paracrystals were observed in uncapped vinblastinetreated cultures but were smaller and more peripherally located in the cytoplasm than in Con A-treated cultures. Evaluation of the percentage of cells capped in Con A/H surface replica preparations revealed that cultures labeled in the presence of colchicine or vinblastine, or in the cold (40), capped as efficiently as cultures processed at 370 (Fig. 7). Treatment of cultures with Con A-FITC resulted in the formation of large juxtanuclear caps on 70-80% of the cells 30 min after labeling at 370 (Fig. 8). The pattern of fluorescence observed is remarkably similar to that obtained after capped cells (Con A treated only) are reacted with FTCCLC (Fig. 9). In separate experiments, comparable numbers of cells displaying the capped fluorescence were observed in Con A-FITC and FTC-CLC labeled cultures. An intensely fluorescent zone was found near the nucleus after incubation with 5 X 10 4 M FTC-CLC. The intense fluorescence associated with caps was absent in uncapped cells processed similarly or in capped cells treated with sodium fluorescein alone. Further, the fluorescence could be diminished by competition with unlabeled colchicine (see Table 1), which is itself not fluorescent under these conditions. A diffuse, granular fluorescence was discernible in the cytoplasm of
4978
Proc. Nat. Acad. Sci. USA 72 (1975)
Cell Biology: Albertini and Clark
.t-..-V
*~.
'..i
S s Out t
FIGS. 1-6. (Legend appears at bottom of following page.)
s
%
St
.
Cell Biology: Albertini and Clark
Proc. Nat. Acad. Sci. USA 72 (1975)
4979
LJ
a)
Uf)
'0 az
LI z
-J
-jT
0
0o
o,0 -CJ
E °
dO
0
U
Li
z V)
w
z 0 U
U
0
0 m
z
-J
0 U
FIG. 7. Cells were capped with Con A and hemocyanin as described in the text, and the percent of capped cells was determined by counting 100 cells per culture on shadow cast replicas. Triplicate cultures were scored; the averages are represented by the bar figures for each of the experimental treatments used.
both capped and uncapped granulosa cells treated with FTC-CLC (Fig. 9). DISCUSSION This study demonstrates that in ovarian granulosa cells the membrane reorganizing event of Con A cap formation elicits an alteration in the structural disposition of cytoplasmic microtubules and in the distribution of colchicine binding proteins. Our results show that microtubules become oriented perpendicularly to the capped portions of the plasma membrane and that vinblastine paracrystals and FTC-CLC are preferentially localized to the capped region of the cell. Analogous systems may exist in conjunction with membrane-microtubule interactions such as (a) establishment of the cleavage furrow in mitosis (14), (b) axoneme growth at the distal (membrane associated) tip of developing flagella (15), (c) phagocytosis (2, 16), (d) viral induced transformation of CHO cells (17), (e) secretion (18), (f) neurite growth (19, 20), and (g) fibroblast locomotion (21), since during each of these events a colchicine-sensitive reorganization of the cytoplasm parallels a modification of the cell surface. The intact microtubules and concentrated FTC-CLC binding observed near the Con A caps demonstrate that the altered membrane domain in cultured granulosa cells affects the intracellular distribution of microtubules and colchicine binding proteins. A large number of observations have shown that microtubules are important in the structural support of cell membranes (22, 23) but the significance of functional interactions between membrane components and cytoplasmic microtubules has only recently received attention (3, 24). It has been shown that Con A-induced capping occurs in fibroblasts transformed by simian virus 40 (8), polymorphonuclear leukocytes (5), and lymphocytes (3, 7, 25) only after exposure to cold or treatment with microtubule disrupting agents. These same treatments appear to abrogate the ability of polymorphonuclear leukocytes to segregate transport sites (1) or to include lectin receptors (2) with phagocytic or interiorizing membrane, suggesting that col-
FIGS. 8 AND 9. Fig. 8. Fluorescence micrograph of a granulosa cell capped with Con A-fluorescein isothiocyanate. X1,150. Fig. 9. A central fluorescent spot is apparent on this cell capped with Con A and subsequently treated with FTC-CLC. X1150.
chicine treatment interrupts the relationship between membrane proteins (24). This implied structural relationship between membranes and intact microtubules prompted the experiments described presently which clearly show an effect of capping on tubulin subunit distribution. The specificity of colchicine binding (9) and vinblastineinduced crystallization (13) were used to indicate localized
FIGS. 1-6 (on preceding page). Light and electron micrographs of Con A capped ovarian granulosa cells. Fig. 1. Phase contrast micrograph of a cell fixed 30 min after treatment with Con A and peroxidase that shows a number of surface protrusions gathered near the cell center. X450. Fig. 2. Corresponding bright-field photograph of the same cell depicted in Fig. 1 that demonstrates the localization of Con Aperoxidase to the centrally located cap. X450. Fig. 3. Surface replica of a cell treated sequentially with Con A and hemocyanin at 370 and fixed 30 min later. The capped area of the cell surface is denoted by the massive collection of hemocyanin molecules in the lower left corner. X6150. (Inset) Individual hemocyanin molecules can be resolved at the edge of the cap; note the irregular surface folds at the cap and the complete clearance of marker molecules from the cell periphery. X21,500. Fig. 4. Transmission electron micrograph of a Con A/H cap 60 min after labeling showing hemocyanin molecules lining surface infoldings and endocytotic vesicles (EV). Many microtubules are visible in longitudinal section underneath the cap. X48,400. Fig. 5. Oblique section through a Con A/H capped portion of a cell illustrating the close association of microtubules (MT) and microfilaments (MF) with the plasma membrane. X51,000. Fig. 6. Electron micrograph of a cell capped with Con A/H and treated with vinblastine for 60 min prior to fixation. A vinblastine induced paracrystal is situated immediately beneath the cap (arrows). X46,200.
4980
Proc. Nat. Acad. Sci. USA 72 (1975)
Cell Biology: Albertini and Clark
concentrations of tubulin. Thus, the formation of vinblastine crystals in the cytoplasm adjacent to the cap and the high density of FTC-CLC bound in the region of the cap suggests that tubulin subunits are localized in sufficient concentrations to produce the cap-associated microtubules. The remarkable increase in the concentration of cap-associated tubulin may indicate that either the capped membrane acts as a preferential organizing site for tubulin or that microtubules are passively pulled into localized regions as a result of Con A-induced surface receptor translocation. Support of the first hypothesis derives from the fact that microtubule assembly from tubulin is concentration dependent (26, 27) and may require activation of tubulin subunits (28, 29) before microtubule assembly. Further, preliminary data suggest that membrane-bound constituents capable of forming a colchicine-sensitive complex with tubulin subunits are present in isolated rabbit polymorphonuclear leukocyte membranes (30). The existence of membrane-bound organizing sites for tubulin is inferred from reports of [14C]colchicine binding to membrane fractions of homogenized liver (31) and the assembly of axoneme microtubules at the distal (membrane associated) tip of elongating flagella (15). Our observations of an increased density of microtubules and FTC-CLC binding in the capped region of cultured granulosa cells lends additional support to the idea that the plasma membrane may serve as an organizing site for tubulin subunits. We thank Drs. Everett Anderson, Thomas Pollard, and David Hamilton for the use of their equipment and Dr. Morris J. Karnovsky for his helpful discussions during various stages of this work and for kindly providing samples of hemocyanin. We also wish to thank Rich Wilkinson for proofreading the manuscript and making many helpful suggestions. These experiments were supported by Training Grant 5 T01 GM00406 from the National Institutes of Health, the U.S. Public Health Service, and a fellowship from the
Pharmaceutical Manufacturer's Association Foundation. 1. Ukena, T. E. & Berlin, R. D. (1972) J. Exp. Med. 136, 1-7. 2. Oliver, J. M., Ukena, T. E. & Berlin, R. D. (1974) Proc. Nat. Acad. Sci. USA 71, 394-398. 3. Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. Acad. Sci. USA 70, 1442-1446. 4. DePetris, S. (1975) J. Cell Biol. 65, 123-146. 5. Oliver, J. M., Zurier, R. B. & Berlin, R. D. (1975) Nature 253, 471-473.
6. Pinto da Silva, P., Martinez-Palomo, A. & -Gonzalez-Robles, A. (1975) J. Cell Biol. 64,538-550. 7. Yahara, I. & Edelman, G. M. (1973) Nature 236, 152-155. 8. Ukena, T. E., Borysenko, J. Z., Karnovsky, M. J. & Berlin, R. D. (1974) J. Cell Biol. 61, 70-82. 9. Wilson, L., Bamburg, J. R., Mizel, S. B., Grisham, L. M. & Creswell, K. M. (1974) Fed. Proc. 33, 158-166. 10. Erickson, G. F., Challis, J. R. G. & Ryan, K. J. (1974) Dev. Biol. 40, 208-224. 11. Ryan, G. B., Borysenko, J. Z. & Karnovsky, M. J. (1974) J. Cell
Biol. 62,351-365. 12. Smith, S. B. & Revel, J. P. (1972) Dev. Biol. 27,434-441. 13. Bensch, K. G. & Malawista, S. E. (1969) J. Cell Biol. 40, 95107. 14. Rappaport, R. (1973) Am. Zool. 13,941-948. 15. Rosenbaum, J. L. & Child, F. M. (1967) J. Cell Biol. 34, 345364. 16. Reaven, E. P. & Axline, S. G. (1973) J. Cell Biol. 59, 12-27. 17. Porter, K. R., Todaro, G. J. & Fonte, V. (1973) J. Cell Biol. 59,
633-642. 18. Stein, O., Sanger, L. & Stein, Y. (1974) J. Cell Biol. 62, 90103. 19. Bunge, M. B. (1973) J. Cell Biol. 56,713-735. 20. Yamada, K. M., Spooner, B. S. & Wessels, N. K. (1970) Proc. Nat. Acad. Sci. USA 66,1206-1210. 21. Vasiliev, Ju. M., Gelfand, I. M., Domnina, L. V., Ivanova, 0. Y., Komm, S. G. & Olshevskaja, L. V. (1970) J. Embryol. Exp. Morphol. 24,625-640. 22. Tilney, L. G. (1971) in Origin and Continuity of Cell Organelles, eds. Reinert, J. & Ursprung, H. (Springer Verlag, Berlin and New York), pp. 222-260. 23. Porter, K. R. (1966) Princ. Biomol. Organ. Ciba Found. Symp., 308-345. 24. Berlin, R. D., Oliver, J. M., Ukena, T. E. & Yin, H. H. (1974) Nature 247,45-46. 25. Yahara, I. & Edelman, G. M. (1973) Exp. Cell Res. 81, 143155. 26. Gaskin, F., Cantor, C. R. & Shelanski, M. L. (1974) J. Mol. Biol. 89,737-758. 27. Inoue, S., Borisy, G. G. & Kiehart, D. P. (1974) J. Cell Biol.
62, 175-184. 28. Shelanski, M. L. (1973) J. Histochem. Cytochem. 21, 529539. 29. Weingarten, M. D., Lockwood, A. H., Hwo, S. & Kirschner, M. W. (1975) Proc. Nat. Acad. Sci. USA 72, 1858-1862. 30. Becker, J. S., Oliver, J. M. & Berlin, R. D. (1975) Nature 254, 152-154. 31. Stadler, J. & Franke, W. W. (1974) J. Cell Biol. 60,297-303.
Cell Biology
Membrane-microtubule interactions: Concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins (cultured ovarian granulosa cells/electron microscopy/vinblastine/fluorescein thiocarbamyl colchicine)
DAVID F. ALBERTINI AND JOHN I. CLARK Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115
Communicated by Don W. Fawcett, September 2, 1975
The relationship between microtubules and concanavalin A surface receptors during concanavalin A cap ping in primary cultures of rabbit ovarian granulosa cells was examined by electron microscopic and fluorescence labeling techniques. Cells treated with concanavalin A and hemocyanin at 40 or 370 and then incubated at 370 for 1 hr formed large juxtanuclear caps that were observed with shadow cast replicas of the cell surface. Thin section analysis of capped cells revealed an abundance of microtubules immediately beneath the cap which were arranged approximately perpendicular to the plane of the membrane. The capping process was unaffected by the antimicrotubule agents colchicine or vinblastine. Further, vinblastine treatment of capped calls resulted in the formation of numerous paracrystals that were confined to the cytoplasm underlying the capped region of the membrane; uncapped cells displayed paracrystals that were randomly dispersed in the cytoplasm. Exposure of fixed cells to fluorescein thiocarbamyl colchicine, which localizes colchicine binding proteins, revealed an intensely fluorescent region that corresponded to the cap; this staining pattern was absent in uncapped cells. These findings indicate that concanavalin A mediated capping modifies the cytoplasmic disposition of microtubules and colchicine binding proteins. Further, it is suggested that the capped region of the plasma membrane is a preferred site of microtubule polymerization. ABSTRACT
of the cytoplasmic microtubule system such that these structures become specifically associated with the capped portion of the plasma membrane. Using a sensitive fluorescent probe for localizing colchicine binding proteins, it was found that tubulin (a colchicine binding protein) is preferentially concentrated in the capped area of the cell. These results suggest that the capped membrane domain may be a preferred site of microtubule polymerization.
MATERIALS AND METHODS Granulosa cells were obtained from preovulatory ovarian follicles (0.9-1.1 mm diameter) of adult nonvirgin Dutch belted rabbits and cultured as described (10). After dissociation of granulosa cells from follicular fluid and eggs by passage through a graded series of micropipettes, cells (from four ovaries) were pooled in fresh medium and cultures seeded by adding four drops, containing approximately 1 X 105 cells, to 18-mm glass coverslips contained in 35 X 10 mm tissue culture dishes. The cells were allowed to attach for 1 hr, after which 2.5 ml of culture medium was added to each dish. The culture medium consisted of McCoy's 5A medium (modified) supplemented with 1% L-glutamine (200 mM stock), 100 units/ml of penicillin, 5 Ag/ml of streptomycin sulfate, and 15% serum obtained from adult female nonvirgin rabbits. Cultures were incubated at 370 in 95% air, 5% CO2; the medium was changed every 2 days. All experiments were performed on 5-day cultures showing predominantly flattened epithelial cell sheets with little cellular overlapping. For thin section and replica preparations, the labeling protocol of Ryan et al. (11) was used. Granulosa cell cultures were washed three times in Earle's Balanced Salt Solution (EBSS) and incubated at 40 or 370 for 1 hr in the presence or absence of colchicine (10-5 M) (Sigma) or vinblastine sulfate (10-5 M) (Eli Lilly Co.). The cells were then exposed to Con A (100 ,.g/ml) (Sigma, grade IV) in EBSS for 10 min at 40 or 370, serially rinsed three times in EBSS, and incubated with either hemocyanin (500 Atg/ml) or horseradish peroxidase (200 ,4g/ml) (Sigma, type VI) in EBSS for 10 min at 40 or 370. After three additional washes with EBSS, the cultures were fixed in 1% glutaraldehyde immediately or after incubation at 370 for 10, 30, or 60 min in EBSS. The inherent distribution of Con A binding sites was determined by labeling cells with Con A and hemocyanin (Con A/H) as above, but after a 10-min fixation in 1% paraformaldehyde in EBSS at 40. The reversibility of Con A binding was evaluated in all experiments by incubating cultures in 0.05 M a-methylD-glucopyranoside (Sigma) for 60 min after the addition of visual markers. In vinblastine and colchicine experiments,
Cytoplasmic microtubules have been implicated in the control of the spatial distribution of membrane transport carriers and lectin receptors in several cell types (1, 2). Treatment of cells with the tetravalent plant lectin concanavalin A (Con A), under appropriate conditions, is known to cause a polar accumulation of receptors which is referred to as a cap. When labeled with low doses of Con A at 370, a variety of cell types, including lymphocytes (3, 4), polymorphonuclear leukocytes from rabbits or beige mice (5), and amebocytes (6), cap spontaneously. Lymphocytes and virally transformed cells are unable to cap in the presence of high doses of Con A at 370. Under these conditions, capping ensues only after treatment with vinca alkaloids, low temperature, or colchicine (3, 7, 8), conditions known to disrupt microtubules (9), suggesting that microtubules participate in the modulation of cell surface receptor mobility. Morphological support for a microtubule-membrane association during capping has been difficult to obtain, largely due to the scarcity of microtubules in the cell types previously studied. We report here that Con A cap formation on ovarian granulosa cells in vitro is accompanied by a reorganization Abbreviations: Con A, concanavalin A; Con A/H concanavalin A by labeling with hemocyanin; EBSS, Earle's Balanced Salt Solution; FITC, fluorescein isothiocyanate; FTCCLC, fluorescein thiocarbamyl colchicine. treatment followed
4976
Proc. Nat. Acad. Sci. USA 72 (1975)
Cell Biology: Albertini and Clark
the drugs were present in all labeling solutions and solutions used after incubation, and in one set of experiments colchicine or vinblastine was added 30 min after incubation and the cultures were fixed 60 min later. For the demonstration of peroxidase activity, cells were reacted with diaminobenzidine (0.5 mg/ml) (Sigma) in 0.1 M Tris buffer (pH 7.4) containing 0.01% H202 for 15 min, washed three times in buffer, and photographed under phase and bright field optics. Phase observations were also made on living cells during the period after incubation. All cultures were post-fixed in 1% aqueous OS04 for 15 min, dehydrated in a cold series of ethanols, and either embedded in an Epon 812-araldite mixture for thin section examination after staining with uranyl acetate and lead, or dried from amyl acetate using a hot air stream to make surface replicas according to the method of Smith and Revel (12). The fluorescein thiocarbamyl colchicine (FTC-CLC) used to localize intracellular colchicine binding proteins was prepared in the following fashion. 1 ml of 10-3 M deacetyl colchicine was dissolved in 95% ethanol and added to 1 ml of an ethanolic solution of 7 X 10-3 M fluorescein isothiocyanate-I (FITC) (Sigma) and was permitted to react at room temperature for 60 min. The FTC-CLC was separated by thin-layer chromatography and eluted from the plates with distilled water to give a final preparation containing 5 X 10-3 M FTC-CLC. This preparation was compared with unlabeled colchicine in a variety of cell types and was found to be identical in all systems tested.* For labeling, cells that had been incubated for 0, 10, 30, and 60 min after treatment with Con A at 370 were fixed with acetone for 5 min at -10° and further incubated for 30 min in 5 X 10-6 M FTC-CLC in 0.14 M phosphate buffer, pH 7.2. In some experiments, cultures were incubated in EBSS for 1 hr, labeled for 10 min in Con A-FITC (10 or 100,ug/ml) (Miles-Yeda) at 370, washed three times with EBSS, incubated for 30 min at 370, and then fixed in acetone for 5 min. All cultures treated with Con A-FITC or FTC-CLC were observed with a Leitz microscope equipped with a Ploem vertical illuminator and fluorescence optics. Photographs were recorded on Kodak tri-X film. The control and experimental treatments using FTC-CLC are depicted in Table 1. RESULTS Observation of living or glutaraldehyde fixed cells 30 min after Con A treatment revealed the presence of numerous surface protrusions localized directly over or adjacent to the nucleus (Fig. 1). Cells labeled with peroxidase for the demonstration of Con A binding sites showed under bright-field optics that this area corresponds to the Con A cap (Fig. 2). Surface replicas of capped cells treated with hemocyanin as a visual marker revealed the presence of a 7- to 12-Am spherical mass of marker molecules generally situated near the cell center (Fig. 3). Close inspection of capped cells showed a complete clearing of hemocyanin molecules from the cell periphery (Fig. 3, inset), with clusters of hemocyanin confined to the cap. a-D-Methyl glucopyranoside-treated cultures showed few hemocyanin molecules on the cell surface, and cultures pretreated with paraformaldehyde prior to Con A labeling displayed a homogeneous distribution of hemocyanin. The capped portion of the granulosa cell surface was highly irregular in contour, and marker molecules were observed bound to coated and uncoated surface invag*
J.
I. Clark and D. Garland, manuscript in preparation.
4977
Table 1. Specificity of FTC-CLC binding to capped and uncapped granulosa cells*
Capped Uncappedt
Controls
FTC-CLC Comp.
0 0
+
+++
+
++
FTC-CLC
Cells were treated with medium lacking FTC-CLC or containing sodium fluorescein alone (Controls). 10-6 M FTC-CLC was competed with 1O-3 M unlabeled colchicine in the same medium (FTC-CLC Comp.). *Fluorescence intensity was arbitrarily scored on a 0 to + + + + basis, where 0 = no apparent fluorescence and ++++ = maximum fluorescence. t Capped cells were treated with Con A (100 gg/ml) for 10 min at 370, washed three times in EBSS, and incubated 30 min at 370 in EBSS before fixation and FTC-CLC treatment as described in the text. Uncapped cells were treated as above except in the absence of Con A.
inations as well as lining more deeply situated tubulovesicular profiles seen in thin sections (Fig. 4). Sections approximately normal to the capped surface revealed an abundance of perpendicularly oriented 24-nm microtubules beneath the capped portion of the plasma membrane. Microtubules were present in untreated granulosa cells but were not as abundant as in Con A-treated cultures and usually were arranged parallel to the long axis of the cell. In granulosa cells actively internalizing Con A caps, hemocyanin-laden vesicles were often seen adjacent to microtubules. In more oblique sections, microtubules and microfilaments (6 and 10 mm in diameter) were observed to course along the cell periphery in close association with the cell membrane (Fig. 5). Cultures capped in the presence of colchicine or vinblastine displayed large, more peripherally located hemocyanin caps that characteristically had smooth contours. Microtubules were rarely observed in drug-treated cultures. The addition of vinblastine to capped cultures (30 min after incubation) resulted in the formation of tubulin paracrystals associated with the capped region of the cell (Fig. 6). The paracrystalline inclusions were comprised of tubular elements having an internal diameter of 20-26 nm, in fair agreement with previously published reports (13). Juxtanuclear paracrystals were observed in uncapped vinblastinetreated cultures but were smaller and more peripherally located in the cytoplasm than in Con A-treated cultures. Evaluation of the percentage of cells capped in Con A/H surface replica preparations revealed that cultures labeled in the presence of colchicine or vinblastine, or in the cold (40), capped as efficiently as cultures processed at 370 (Fig. 7). Treatment of cultures with Con A-FITC resulted in the formation of large juxtanuclear caps on 70-80% of the cells 30 min after labeling at 370 (Fig. 8). The pattern of fluorescence observed is remarkably similar to that obtained after capped cells (Con A treated only) are reacted with FTCCLC (Fig. 9). In separate experiments, comparable numbers of cells displaying the capped fluorescence were observed in Con A-FITC and FTC-CLC labeled cultures. An intensely fluorescent zone was found near the nucleus after incubation with 5 X 10 4 M FTC-CLC. The intense fluorescence associated with caps was absent in uncapped cells processed similarly or in capped cells treated with sodium fluorescein alone. Further, the fluorescence could be diminished by competition with unlabeled colchicine (see Table 1), which is itself not fluorescent under these conditions. A diffuse, granular fluorescence was discernible in the cytoplasm of
4978
Proc. Nat. Acad. Sci. USA 72 (1975)
Cell Biology: Albertini and Clark
.t-..-V
*~.
'..i
S s Out t
FIGS. 1-6. (Legend appears at bottom of following page.)
s
%
St
.
Cell Biology: Albertini and Clark
Proc. Nat. Acad. Sci. USA 72 (1975)
4979
LJ
a)
Uf)
'0 az
LI z
-J
-jT
0
0o
o,0 -CJ
E °
dO
0
U
Li
z V)
w
z 0 U
U
0
0 m
z
-J
0 U
FIG. 7. Cells were capped with Con A and hemocyanin as described in the text, and the percent of capped cells was determined by counting 100 cells per culture on shadow cast replicas. Triplicate cultures were scored; the averages are represented by the bar figures for each of the experimental treatments used.
both capped and uncapped granulosa cells treated with FTC-CLC (Fig. 9). DISCUSSION This study demonstrates that in ovarian granulosa cells the membrane reorganizing event of Con A cap formation elicits an alteration in the structural disposition of cytoplasmic microtubules and in the distribution of colchicine binding proteins. Our results show that microtubules become oriented perpendicularly to the capped portions of the plasma membrane and that vinblastine paracrystals and FTC-CLC are preferentially localized to the capped region of the cell. Analogous systems may exist in conjunction with membrane-microtubule interactions such as (a) establishment of the cleavage furrow in mitosis (14), (b) axoneme growth at the distal (membrane associated) tip of developing flagella (15), (c) phagocytosis (2, 16), (d) viral induced transformation of CHO cells (17), (e) secretion (18), (f) neurite growth (19, 20), and (g) fibroblast locomotion (21), since during each of these events a colchicine-sensitive reorganization of the cytoplasm parallels a modification of the cell surface. The intact microtubules and concentrated FTC-CLC binding observed near the Con A caps demonstrate that the altered membrane domain in cultured granulosa cells affects the intracellular distribution of microtubules and colchicine binding proteins. A large number of observations have shown that microtubules are important in the structural support of cell membranes (22, 23) but the significance of functional interactions between membrane components and cytoplasmic microtubules has only recently received attention (3, 24). It has been shown that Con A-induced capping occurs in fibroblasts transformed by simian virus 40 (8), polymorphonuclear leukocytes (5), and lymphocytes (3, 7, 25) only after exposure to cold or treatment with microtubule disrupting agents. These same treatments appear to abrogate the ability of polymorphonuclear leukocytes to segregate transport sites (1) or to include lectin receptors (2) with phagocytic or interiorizing membrane, suggesting that col-
FIGS. 8 AND 9. Fig. 8. Fluorescence micrograph of a granulosa cell capped with Con A-fluorescein isothiocyanate. X1,150. Fig. 9. A central fluorescent spot is apparent on this cell capped with Con A and subsequently treated with FTC-CLC. X1150.
chicine treatment interrupts the relationship between membrane proteins (24). This implied structural relationship between membranes and intact microtubules prompted the experiments described presently which clearly show an effect of capping on tubulin subunit distribution. The specificity of colchicine binding (9) and vinblastineinduced crystallization (13) were used to indicate localized
FIGS. 1-6 (on preceding page). Light and electron micrographs of Con A capped ovarian granulosa cells. Fig. 1. Phase contrast micrograph of a cell fixed 30 min after treatment with Con A and peroxidase that shows a number of surface protrusions gathered near the cell center. X450. Fig. 2. Corresponding bright-field photograph of the same cell depicted in Fig. 1 that demonstrates the localization of Con Aperoxidase to the centrally located cap. X450. Fig. 3. Surface replica of a cell treated sequentially with Con A and hemocyanin at 370 and fixed 30 min later. The capped area of the cell surface is denoted by the massive collection of hemocyanin molecules in the lower left corner. X6150. (Inset) Individual hemocyanin molecules can be resolved at the edge of the cap; note the irregular surface folds at the cap and the complete clearance of marker molecules from the cell periphery. X21,500. Fig. 4. Transmission electron micrograph of a Con A/H cap 60 min after labeling showing hemocyanin molecules lining surface infoldings and endocytotic vesicles (EV). Many microtubules are visible in longitudinal section underneath the cap. X48,400. Fig. 5. Oblique section through a Con A/H capped portion of a cell illustrating the close association of microtubules (MT) and microfilaments (MF) with the plasma membrane. X51,000. Fig. 6. Electron micrograph of a cell capped with Con A/H and treated with vinblastine for 60 min prior to fixation. A vinblastine induced paracrystal is situated immediately beneath the cap (arrows). X46,200.
4980
Proc. Nat. Acad. Sci. USA 72 (1975)
Cell Biology: Albertini and Clark
concentrations of tubulin. Thus, the formation of vinblastine crystals in the cytoplasm adjacent to the cap and the high density of FTC-CLC bound in the region of the cap suggests that tubulin subunits are localized in sufficient concentrations to produce the cap-associated microtubules. The remarkable increase in the concentration of cap-associated tubulin may indicate that either the capped membrane acts as a preferential organizing site for tubulin or that microtubules are passively pulled into localized regions as a result of Con A-induced surface receptor translocation. Support of the first hypothesis derives from the fact that microtubule assembly from tubulin is concentration dependent (26, 27) and may require activation of tubulin subunits (28, 29) before microtubule assembly. Further, preliminary data suggest that membrane-bound constituents capable of forming a colchicine-sensitive complex with tubulin subunits are present in isolated rabbit polymorphonuclear leukocyte membranes (30). The existence of membrane-bound organizing sites for tubulin is inferred from reports of [14C]colchicine binding to membrane fractions of homogenized liver (31) and the assembly of axoneme microtubules at the distal (membrane associated) tip of elongating flagella (15). Our observations of an increased density of microtubules and FTC-CLC binding in the capped region of cultured granulosa cells lends additional support to the idea that the plasma membrane may serve as an organizing site for tubulin subunits. We thank Drs. Everett Anderson, Thomas Pollard, and David Hamilton for the use of their equipment and Dr. Morris J. Karnovsky for his helpful discussions during various stages of this work and for kindly providing samples of hemocyanin. We also wish to thank Rich Wilkinson for proofreading the manuscript and making many helpful suggestions. These experiments were supported by Training Grant 5 T01 GM00406 from the National Institutes of Health, the U.S. Public Health Service, and a fellowship from the
Pharmaceutical Manufacturer's Association Foundation. 1. Ukena, T. E. & Berlin, R. D. (1972) J. Exp. Med. 136, 1-7. 2. Oliver, J. M., Ukena, T. E. & Berlin, R. D. (1974) Proc. Nat. Acad. Sci. USA 71, 394-398. 3. Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. Acad. Sci. USA 70, 1442-1446. 4. DePetris, S. (1975) J. Cell Biol. 65, 123-146. 5. Oliver, J. M., Zurier, R. B. & Berlin, R. D. (1975) Nature 253, 471-473.
6. Pinto da Silva, P., Martinez-Palomo, A. & -Gonzalez-Robles, A. (1975) J. Cell Biol. 64,538-550. 7. Yahara, I. & Edelman, G. M. (1973) Nature 236, 152-155. 8. Ukena, T. E., Borysenko, J. Z., Karnovsky, M. J. & Berlin, R. D. (1974) J. Cell Biol. 61, 70-82. 9. Wilson, L., Bamburg, J. R., Mizel, S. B., Grisham, L. M. & Creswell, K. M. (1974) Fed. Proc. 33, 158-166. 10. Erickson, G. F., Challis, J. R. G. & Ryan, K. J. (1974) Dev. Biol. 40, 208-224. 11. Ryan, G. B., Borysenko, J. Z. & Karnovsky, M. J. (1974) J. Cell
Biol. 62,351-365. 12. Smith, S. B. & Revel, J. P. (1972) Dev. Biol. 27,434-441. 13. Bensch, K. G. & Malawista, S. E. (1969) J. Cell Biol. 40, 95107. 14. Rappaport, R. (1973) Am. Zool. 13,941-948. 15. Rosenbaum, J. L. & Child, F. M. (1967) J. Cell Biol. 34, 345364. 16. Reaven, E. P. & Axline, S. G. (1973) J. Cell Biol. 59, 12-27. 17. Porter, K. R., Todaro, G. J. & Fonte, V. (1973) J. Cell Biol. 59,
633-642. 18. Stein, O., Sanger, L. & Stein, Y. (1974) J. Cell Biol. 62, 90103. 19. Bunge, M. B. (1973) J. Cell Biol. 56,713-735. 20. Yamada, K. M., Spooner, B. S. & Wessels, N. K. (1970) Proc. Nat. Acad. Sci. USA 66,1206-1210. 21. Vasiliev, Ju. M., Gelfand, I. M., Domnina, L. V., Ivanova, 0. Y., Komm, S. G. & Olshevskaja, L. V. (1970) J. Embryol. Exp. Morphol. 24,625-640. 22. Tilney, L. G. (1971) in Origin and Continuity of Cell Organelles, eds. Reinert, J. & Ursprung, H. (Springer Verlag, Berlin and New York), pp. 222-260. 23. Porter, K. R. (1966) Princ. Biomol. Organ. Ciba Found. Symp., 308-345. 24. Berlin, R. D., Oliver, J. M., Ukena, T. E. & Yin, H. H. (1974) Nature 247,45-46. 25. Yahara, I. & Edelman, G. M. (1973) Exp. Cell Res. 81, 143155. 26. Gaskin, F., Cantor, C. R. & Shelanski, M. L. (1974) J. Mol. Biol. 89,737-758. 27. Inoue, S., Borisy, G. G. & Kiehart, D. P. (1974) J. Cell Biol.
62, 175-184. 28. Shelanski, M. L. (1973) J. Histochem. Cytochem. 21, 529539. 29. Weingarten, M. D., Lockwood, A. H., Hwo, S. & Kirschner, M. W. (1975) Proc. Nat. Acad. Sci. USA 72, 1858-1862. 30. Becker, J. S., Oliver, J. M. & Berlin, R. D. (1975) Nature 254, 152-154. 31. Stadler, J. & Franke, W. W. (1974) J. Cell Biol. 60,297-303.
