EXPERIMENTAL
CELL
RESEARCH
200,361-369
(1992)
Attachment of Mitochondria to Intermediate Filaments in Adrenal Cells: Relevance to the Regulation of Steroid Synthesis’ GHANIM ALMAHBOBI, LINDY J. WILLIAMS, AND PETER F. HALLS Department
of Endocrinology,
Prince
of Wales
Hospital,
The rate of steroid synthesis is regulated by the rate of transport of cholesterol from lipid droplets to mitochondria. We have previously demonstrated that lipid droplets in adrenal cells are tightly attached to intermediate filaments. Here we now show that mitochondria colocalize with intermediate filaments in modified double indirect immunofluorescence and by electron microscopy of extracted adrenal cells. Direct contact between mitochondria and intermediate filaments was established by examination of stereo pairs of electron micrographs from extracted cells. The attachment of both droplets and mitochondria to intermediate filaments suggests possible mechanisms for this form of intracellular transport of cholesterol to mitochondria and hence for the regulation of steroid synthesis. o issz ACTdemic
Press,
Inc.
INTRODUCTION
It has been known for some time that the cholesterol used by steroidogenic cells for the synthesis of steroid hormones is stored in the cytoplasm-much of it in the conspicuous lipid droplets that form a distinctive feature of these cells [l-5]. On the other hand the conversion of cholesterol to steroid hormones begins with a reaction catalyzed by a cytochrome P450 in the inner mitochondrial membrane (P45Oscc) [6]. This enzymatic reaction, called side-chain cleavage of cholesterol (see), results in the formation of the key intermediate pregnenolone. Clearly cholesterol must move from cytoplasm to mitochondria to enter the steroidogenic pathway. It turns out that this process of intracellular transport of cholesterol is the rate-limiting step in the pathway and is specifically stimulated by the trophic hormones ACTH and LH. This step requires the cytoskeleton and, in particular, actin [7-91. In investigating the transport of cholesterol in adrenal cells we found that lipid droplets containing cholesterol ester are attached to intermediate filaments and i Supported * To whom
by NH&MRC reprint requests
Grant 900706. should be addressed.
Randwick,
2031 New
South
Wales,
Australia
remain associated with these filaments after severe extraction with Triton X-100 [4, 51. The contents of the droplets appear to be protected by a capsule containing vimentin [4, 51. To pursue these investigations further we attempted to obtain a reproducible preparation of cytoskeleton in which both lipid droplets and mitochondria are present with the cytoskeleton so that the movement of droplets to mitochondria can be investigated in vitro. For these studies we used both Y-l adrenal tumor cells and primary cultures of bovine fasciculata cells. MATERIALS
AND METHODS
Cell preparation and culture. Primary cultures of bovine fasciculata cells were prepared as described elsewhere [5, lo]. Y-l mouse adrenal tumor cells (American Type Culture) were grown in culture as described elsewhere [4,5,7]. Both cell types were plated at a density of 1 X lOa cells per milliliter in Ham’s F12 plus Dulbecco’s MEM with 12.5% (v/v) horse serum and 2.5% (v/v) fetal calf serum. Production of steroids (20adihydroprogesterone for Y-l cells and cortisol for beef cells) was measured by radioimmunoassays [7, 111 on samples of incubation medium. Cells were grown on glass coverslips, slide chambers, or plastic dishes for morphological studies and in plastic dishes for biochemical assays. Where indicated, experiments were performed in the above medium without serum (serum-free medium). Rupture and extraction of cells. (i) Cells were broken by osmotic lysis using the method of Beckers andcollaborators [12]. (ii) Homogenization was carried out by scraping cells from plastic dishes into a glass-glass Dounce homogenizer in buffer. Cells were ruptured by 20 strokes. (iii) Mild extraction was performed by standing cells in 0.5% Triton X-100 for 5 min as previously described for preparation of cytoskeleton [4, 131. Severe extraction used 1% Triton for 10 minthis procedure was performed once or twice on the same cells as described with individual experiments. To prepare intermediate filaments 1% Triton for 10 min once only was followed by ammonium sulfate [13] in 1% Triton for a further 10 min. Fluorescence microscopy. Y-l and bovine fasciculata cells were grown on glass coverslips and were incubated with serum-free medium containing Rhodamine 123 (10 pglml) for 30 min at 37°C before examination by fluorescence microscopy [14]. In some studies preparations of intermediate filaments were treated with Nile red to stain lipid droplets [4]. Zmmunofluorescence microscopy. This procedure was performed on cells grown on glass slide chambers. The cells were extracted by the mild procedure, by severe extraction, by severe extraction twice, and by treatment with ammonium sulfate to prepare intermediate filaments. (a) Single indirect immunofluorescence was performed to display intermediate filaments or mitochondria exactly as described previously [4].
361
0014-4827t92
Copyright All
rights
0
1992 of reproduction
by Academic in eny
form
$5.00
Press, Inc. reserved.
362
ALMAHBOBI,
WILLIAMS,
AND HALL
MITOCHONDRIA
AND
INTERMEDIATE
FILAMENTS
363
FIG. 1. Fluorescence microscopy of living adrenal cells. Cultured cells were incubated with rhodamine 123 to stain mitochond ria (10 ng ml-‘) for 30 min. (A) Y-l cells and (B) bovine fasciculata. n, nucleus; bar, 2 pm. FIG. 2. Double indirect immunofluorescence microscopy of mildly extracted adrenal cells. Cells were subjected to mild extras cti4 on (see Materials al nd Methods). (A and B) Y-l cells and (C! and D) bovine fasciculata cells. Each pair of panels (A and B; C and D) shows th e same cells at two (excitation wavelengths. Cells were stained for both mitochondria (A and C) and intermediate filaments (B and D). n, nucll E2lE S;bar, 5 w-n. FIG. 3. Double indirect immunofluorescence of the cytoskeleton of Y-l cells. Mitochondria and intermediate filaments were smtai ned by the 43 proc’ edure described under Materials and Methods so that both mitochondria and intermediate filaments can be seen at one f?XC :itation wave length I. n, nucleus; bar, 2.5 pm. FIG. 4. Double indirect immunofluorescence of intermediate filaments. Intermediate filaments prepared from Y-l cells were sts lint sd with anti-cytochl rome oxidase (A), anti-vimentin (B), or anti-vimentin and Nile red (C). Arrows, lipid droplets; n, nucleus; bar, (A) and(B) 51 m, (Cl 2 pm.
364
FIG. 5. Electron thin sections. Arrows tus; m, mitochondrion;
ALMAHBOBI,
microscopy of bovine indicate intermediate bar, 0.125 pm.
fasciculata filaments
WILLIAMS,
AND
HALL
cells. Cells were subjected to lysis (A) or homogenization (B) followed in direct contact with mitochondria. er, dilated endoplasmic reticulum;
(b) Double indirect immunofluorescence microscopy used the standard procedure with four different antibodies raised in four species. The four antibodies were applied separately in the following order: The first antibody for mitochondria was rabbit anti-cytochrome oxidase (kindly provided by Dr. E. Racker, Department of Biochemistry, Cornell University, Ithaca, NY) diluted 1:30. Incubation was for 60 min at room temperature. The second antibody was donkey anti-rabbit IgG conjugated to Texas red diluted 1:40 for 60 min. Intermediate filaments were stained with goat anti-vimentin diluted 1:20 for 60 min and was followed by swine anti-goat IgG conjugated to FITC diluted 1:20 for 30 min. (c) The modified procedure (4:3 immunofluorescence, i.e., four anti-
by preparation of g, Golgi appara-
bodies raised in three species) has been reported in detail [23]. The first three antibodies are the same as those in (b) and the fourth antibody is rabbit anti-goat conjugated to FITC diluted 1:40 for 30 min. In this procedure we substitute rabbit anti-goat IgG conjugated to FITC for the corresponding swine IgG with the result that the new antibody (rabbit anti-goat IgG) will recognize goat anti-vimentin and be recognized by donkey anti-rabbit IgG. Since the latter antibody is indirectly attached to mitochondria (cytochrome oxidase), these structures will be stained with both Texas red and FITC. As a result it is possible to examine both mitochondria and intermediate filaments in the same image (in this case with the blue filter) without the need to change filters and compare two images. At the same time one of
MITOCHONDRIA
AND
INTERMEDIATE
365
FILAMENTS
F ‘IG. 6. Stereo pairs of electronmicrographs of bovine fasciculata cells were prepared. The preparations and thil n sections of the monolayer inte !rrnt diate filaments in direct contact with mitochondria (m). Bar,
cells subjected to lysis. Bovine fasciculata cells were subjected were tilted at -12’ (A) and +12’ (B) and photographed. Arror 0.09 em.
these structures, namely mitochondria, could be identified alone by using the green filter. Other details have been described elsewhere [23]. Electron microscopy. Cells subjected to lysis were processed as monolayers. Homogenized and extracted cells were handled as pellets after scraping from dishes. Samples were fixed in a one-half concentration of Karnovsky fixative for 60 min as described previously [3,5]. Specimens were embedded in Spurr’s resin. Some experiments were performed by the method of Franke et al. using osmium tetroxideglutaraldehyde fixation [15]. Thin sections were examined under a Hitachi 7000 electron microscope. Stereo pairs were prepared by tilting the microscope stage. Analytical procedures. Protein was measured by the method of Bradford [16]. Cytochrome-c oxidase was measured by a published spectrophotometric method [17]. This method is based upon the rate of oxidation of ferrocytochrome c by following the decrease in absorbance at 550 nm. Side-chain cleavage activity was measured as the conversion of cholesterol to pregnenolone. Extracted cells are incubated at 37°C with 10 nM cholesterol, 3.5 mg/ml adrenodoxin reductase, 2 mg/ml adrenodoxin in 50 mM phosphate buffer, pH 7.4. The reaction is started by the addition of 8 mg/ml NADPH. The electron carriers adrenodoxin and its reductase pass electrons from NADPH to the P450 side-chain cleavage enzyme to permit the enzyme to convert cholesterol to pregnenolone (side-chain cleavage of cholesterol). Cholesterol was measured by radioimmunoassay as described by
Mrotek and Hall [7]. The minimal amount of pregnenolone by this method is 0.01 pmol/min/mg protein. Materials. Sources of all materials used were the same described previously [4, 51 except where indicated above.
lysis
detected as those
RESULTS
Fluorescence Microscopy
of Living
Adrenal
Cells
Figure 1 shows fluorescence microscopy of Y-l and bovine fasciculata cells in culture using rhodamine 123 to stain mitochondria in living cells. In both cell types mitochondria appear as filamentous structures but those from bovine fasciculata are considerably more elongated than those from Y-l cells. These organelles are concentrated in the perinuclear region of both cell types.
Immunofluorescence (1) Cytoskeleton. extracted
with
Triton
of Extracted Adrenal X-100
Adrenal Cells
cells of both types were (0.5% v/v for 5 min) and
366
ALMAHBOBI,
WILLIAMS,
AND HALL
FIG. 7. Stereo pairs of electronmicrographs of Y-l cells. Cells were extracted with Triton X-100 using mild conditions (see Materials and Method Is). Thin sections of the cells were tilted to -6” (Al and +6’ (B) and photographed. Arrows indicate intermediate filaments in direct contact with mitochondria (m). n, nucleus; bar, 0.08 pm.
subjected to indirect double immunofluorescence staining using rabbit anti-cytochrome oxidase and donkey anti-rabbit IgG conjugated to Texas red, followed by goat anti-vimentin and swine anti-goat IgG conjugated to FITC, i.e., four antibodies raised in four species (Figs. 2A-2D). It is clear that mitochondria are retained in the cell during mild extraction since intense staining of cytochrome oxidase is seen in punctate structures, especially in the perinuclear region (Figs. 2A and 2C). Staining appears to be confined to mitochondria. Intermediate filaments are clearly revealed by the antibody conjugated to FITC (Figs. 2B and 2D). In order to examine the relative distributions of mitochondria and intermediate filaments we repeated this procedure in a modified form, which we refer to as 4~3 immunofluorescence, as described under Materials and Methods. The intense yellowish green color of the mitochondria seen with the blue filter is quite distinct from the green color of the filaments (Fig. 3). Mitochondria are also distinguishable from intermediate filaments by
morphology. With the green filter only mitochondria appear red as shown in Figs. 2A and 2C (not shown). It is clear that mitochondria are closely associated with and occur along the course of the vimentin filaments. Similar findings were made with bovine fasciculata cells (not shown). (II) Intermediate filaments. Y-l cells were extracted and treated with ammonium sulfate to prepare intermediate filaments (see Materials and Methods) and the filaments were subjected to indirect double immunofluorescence staining as in Fig. 2 (four antibodies raised in four species). Mitochondria are not visible as such but patches of red staining can be seen. The procedure used has destroyed mitochondria but these residual fragments of stained material can be seen (Fig. 4A). Intermediate filaments are clearly outlined as before with the aid of anti-vimentin and second antibody (Fig. 4B). Similar findings are seen with bovine fasciculata cells (not shown). Finally by using anti-vimentin with a second antibody and the lipid stain Nile red, we see that
MITOCHONDRIA
AND
INTERMEDIATE
FIG. 8. Stereo pairs of bovine fasciculata cells mildly extracted with Triton. were prepared. Sections were tilted to -6” (A) and +6’ (B) and photographed. mitochondria (m). n, nucleus; bar, 0.07 pm.
in the intermediate filament preparation, lipid droplets are to be seen in keeping with our earlier reports [4, 51 (Fig. 4C). (III) Severe extraction. When cells of either type are subjected twice to severe extraction (see Materials and Methods) mitochondria are entirely removed from the preparation. Evidently the removal of mitochondria is not specifically related to the use of ammonium sulfate (data not shown). Electron
Microscopy
Bovine fasciculata cells were broken by two procedures designed to produce as little disruption of cellular architecture as possible, namely osmotic lysis and gentle homogenization. A thin section of a cultured bovine fasciculata cell subjected to lysis is shown in Fig. 5A. In addition to the usual cellular structures, lo-nm filaments are seen in contact with mitochondria. A similar association is seen in thin sections of homogenized Y-l cells (Fig. 5B). In order to examine contact points between filaments
FILAMENTS
367
Cells were extracted by the mild procedure and thin sections Arrows indicate intermediate filaments in direct contact with
and mitochondria, stereo pairs were made by tilting specimens. Figure 6 shows a thin section of a bovine fasciculata cell subjected to lysis. Intermediate filaments are clearly revealed and well-preserved mitochondria are seen with filaments passing between two of these organelles. Figures 7 and 8 show stereo pairs of thin sections of cytoskeletons from Y-l and bovine fasciculata cells, respectively. Careful examination of the stereo pairs shows that direct contact occurs between intermediate filaments and mitochondria in cells of both types. Furthermore preparation of intermediate filaments, which involves an ammonium sulfate treatment, removes all mitochondria (not shown). These findings are consistent with those described above for immunofluorescence microscopy. Characterization
of Mitochondria
Table 1 shows that the preparation of cytoskeleton (mild extraction) leaves significant amounts of cytochrome oxidase activity in the extracted cells-both Y-l and fasciculata. The need for Triton to reveal maxi-
368
ALMAHBOBI,
TABLE Cytochrome
WILLIAMS,
1
Oxidase
in Adrenal
Cells
Activity (nmol/min/lO’
cells)
Treatment
Cell Y-l Bovine fasciculata
Homogenate
Homogenate and Triton x-100
Mild extraction
19
102
49
18
65
43
Intermediate filaments 12 0.8
Note. Adrenal cells were homogenized and aliquots were taken for measurement of cytochrome oxidase. The homogenate was then treated with Triton X-100 (v/v) and aliquots were again taken for assay of cytochrome oxidase activity. Other cells were subjected to mild extraction or used to prepare intermediate filaments. Cytochrome oxidase activity was measured on the cell residues. These data were a result of two separate experiments and the figures are rounded to the closest values.
ma1 activity in the homogenate makes it difficult to establish an exact recovery of cytochrome oxidase. However, approximately half of this enzyme activity remains after mild extraction. The intermediate filaments have lost most of the cytochrome oxidase. The small amount seen in the filament preparations is consistent with faint mitochondrial staining seen in the immunofluorescence studies shown in Fig. 4A. In other experiments we found that the mitochondria of mildly extracted cells are still capable of catalyzing side-chain cleavage of cholesterol, albeit at a greatly reduced rate compared to that of mitochondria prepared from homogenized cells (1.5 and 0.2 pmol of pregnenolone/min/mg protein in Y-l and bovine fasciculata cells, respectively). These values are significantly greater than background (0.01 pmol/min/mg protein). The mitochondria of the extracted cells require addition of electron carriers to catalyze side-chain cleavage since these proteins are readily removed from mitochondria. DISCUSSION
The two important findings to emerge from these studies are first that we can prepare an adrenal cell extract from which cytosol, plasma membrane, and much of the internal structure of the cell have been removed, leaving no more than cytoskeleton, lipid droplets, and mitochondria, and second the demonstration that in such extracted adrenal cells functional mitochondria are attached to intermediate filaments. Since the ratelimiting step in steroid synthesis-one that is stimulated by ACTH and its second messenger cyclic AMPinvolves the transport of cholesterol from lipid droplets
AND HALL
to mitochondria, it would be helpful to use a preparation of cells that contains these two structures. Such apreparation should be permeable to large molecules and it would be desirable to remove most of the remaining cell structure. With such a preparation, extracts, molecules, and organelles could be added back to restore the process of intracellular transport of cholesterol. It is possible to measure transport of cholesterol to mitochondria and the effect of ACTH on this process in intact adrenal cells [7-91. Experimental approaches to transport of cholesterol would be facilitated by using the simplest subcellular system that contains the two structures involved, namely lipid droplets and mitochondria. In searching for such a system we discovered that lipid droplets containing cholesterol ester are very tightly attached to intermediate filaments [4,5]. We present here a mild extraction procedure for adrenal cells that leaves all three structures, i.e., droplets, filaments, and mitochondria, in the extracted cell. The conditions used produce sufficient clearing of cell contents to permit thorough morphological studies with light and electron microscopy. Moreover the mitochondria are capable of performing the side-chain cleavage of cholesterol. In addition the extracted cell contains approximately onehalf of the original cellular cytochrome oxidase. The evidence for attachment of mitochondria to intermediate filaments includes localization in fluorescence microscopy and direct visualization of contact points on electron microscopy. Although the resolution of fluorescence microscopy does not allow visualization of contact points, the distribution of individual mitochondria with individual filaments seen in our preparations indicates colocalization of these two structures. In view of the repeated washing of cells involved in fluores-
cence microscopy the mitochondrial membrane must be attached to some internal structure. Moreover when we compare intact living cells with mildly extracted cells we see no change in the distribution of mitochondria. Although the retention of mitochondria following mild extraction does not in itself indicate binding of these structures to intermediate filaments, all the above considerations together with the evidence for direct contact seen on electron microscopy make it clear that mitochondria are firmly bound to intermediate filaments. Since more than 50% of a mitochondrial marker enzyme is retained on mild extraction, it. is very unlikely that the mitochondria are removed from their normal intracellular location to become artifictitiously attached to the intermediate filaments. Moreover during mild extraction some mitochondrial fragments remain clearly visible in fluorescence microscopy, indicating tight binding of these organelles to intermediate filaments. It would appear that some form of attachment must be necessary to hold the fragments within the disrupted cell. Finally the scattered patches of mitochondrial staining seen
MITOCHONDRIA
AND INTERMEDIATE
after severe extraction suggest the possibility that fragments of mitochondria remain bound to intermediate filaments. These filaments are virtually the only structures present after severe extraction which removes actin and tubulin from the cell [4]. Mitochondria remaining after mild extraction are morphologically and biochemically damaged, appearing as small vesicular structures which are however still capable of catalyzing the side-chain of cholesterol. It is also of interest to note that the two cell types show certain differences on extraction. For example, the fasciculata cells appear to suffer greater morphological damage for the same intensity of extraction. It is however useful to be able to prepare extracted cells from both a cell line and primary cultures of normal cells since both Y-l [ 181 and bovine fasciculata cells have been widely used in studies of adrenal cell function and because of important differences between cytoskeletons of normal and transformed cells. Attachment of mitochondria to intermediate filaments has been reported for other cell types [19, 201. In skin fibroblasts a protein from intermediate filaments called lEF24 is found to be closely associated with mitochondria [ 191. Clearly it will be important to determine the mechanism of attachment of these two structures in adrenal cells. In the meantime it is important to note that the association of mitochondria with intermediate filaments reported here is more than a surface contact between these structures. The components of inner membrane (side-chain cleavage enzyme and cytochrome oxidase) are attached to the filaments, indicating an intimate binding between intramitochondrial enzymes and intermediate filaments. The intermediate filaments seen in Figs. 2B and 2D show a punctate appearance that we interpret as further evidence of the intimate association between mitochondrial contents and intermediate filaments. This striped appearance (which is seen only where mitochondria are present) probably represents a change in the organization of vimentin where the filaments are in contact with mitochondria. However, we cannot exclude some leakage of fluorescent signal in the FITC channel. The observation that both functional mitochondria and cholesterol-containing lipid droplets are attached to intermediate filaments, together with earlier studies by other workers showing lipid droplets in intimate contact with mitochondria (so-called docking) [21], suggests possible mechanisms whereby the vectorial characteristics of intermediate filaments could facilitate targeting of droplets to mitochondria. Previous studies from this laboratory showed that microfilaments are involved in the increase in the transport of cholesterol to mitochondria in response to ACTH and cyclic AMP [ 7Received Revised
October 3,199l version received
February
12, 1992
369
FILAMENTS
91. This may point to a contractile mechanism which could act in concert with the directional properties of intermediate filaments to facilitate transport of droplets to mitochondria or vice versa. The idea is made attractive by recent studies showing that in permeabilized fibroblasts ATP promotes reorganization of intermediate filaments as the result of contraction of peripheral actomyosin [22]. The authors are grateful to Dr. Adrian Zammit for the assays of cytochrome oxidase and side-chain cleavage and to Eve Smith for typing the manuscript. We are also grateful to the staff of the Electron Microscopy Unit at the University of New South Wales and to the Director, Dr. M. R. Dixon.
REFERENCES 1. 2.
Rhodin, J. A. G. (1971) J. UZtrastruct. Res. 34, 23-71. Almahbobi, G., Silberzahn, N., Fakhri, R., and Silberzahn, (1985) Arch. Anat. Microsc. Morphol. Exp. 74, 193-203.
3.
Almahbobi, G., Papadopoulos, V., Carreau, P. (1988) Biol. Reprod. 38,653-665.
4. 5.
Almahbobi, Almahbobi,
6.
Hall, Ed.),
G., and Hall, G., Williams,
P.
S., and Silberzahn,
P. F. (1990) J. Cell Sci. 97, 679-687. J. L., and Hall, P. F. (1992) J. Cell Sci.
101,383-393. P. F. (1987) in Hormonal Proteins and Peptides (Li, C. H., Vol. XIII, pp. 89-125, Academic Press, New York. J. J., and Hall,
Mrotek,
Hall, P. F., Charponnier, C., Nakamura, (1979) J. Biol. Chem. 254,9080-9084.
M.,
9.
Osawa, 1342.
J. Cell Biol.
S., Betz,
P. F. (1977)
G., and Hall,
Biochemistry
16,3177-3181.
I. a.
P. F. (1984)
10.
Gospodarowicz, D., Ill, C. R., Hornsby, (1977) Endocrinology 100, 1080-1089.
11.
Yanagibashi, K., Ohno, Y., Kawamura, (1988) Endocrinology 123, 2075-2082. Beckers, C. J. M., Keller, D. S., and Balch, 523-534.
12. 13.
Fey, E. G., Wan, 1973-1984.
14.
Johnson, L. V., Walsh, M. L., and Chen, Acad. Sci. USA 77,990-994.
15.
Franke, Keenan, Bradford,
16.
K. M.,
and Penman,
and Gabbiani, 99,1335-
P. J., and M.,
and
Gill,
G. N.
Hall,
P. F.
W. E. (1987)
S. (1984)
G.
Cell 50,
J. Cell Biol.
L. B. (1980)
W. W., Ludez, M. R., Kartenbeck, J., Zerban, T. W. (1976) J. Cell Biol. 69,173-195. M. (1976) Anal. Biochem. 72, 248-254.
98,
Proc. Natl. H., and
17.
Wharton, D. C., and Tzagoloff, A. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., Eds.), Vol. 10, pp. 245-250, Academic Press, San Diego.
18.
Kowal, J. (1970) Recent Prog. Horm. Res. 26,623-677. Mose-Larsen, P., Bravo, R., Fey, S. J., Small, J. V., and Celis, J. E. (1982) Cell 31,681-692. Lin, A., Krockinglic, G., and Penman, S. (1990) Proc. Natl. Acad. Sci. USA 87,8565-8569.
19. 20.
119, 611-618.
21.
Merry,
22.
Tint, I. S., Hollenbeck, P. J., Verkhovsky, A. B., Surgucheva, J. F., and Bershadsky, A. D. (1991) J. Cell Sci. 98, 375-384. Almahbobi, G., and Hall, P. F. (1992) Hi&o&em. J., in press.
23.
B. J. (1975)
J. Anat.
CELL
RESEARCH
200,361-369
(1992)
Attachment of Mitochondria to Intermediate Filaments in Adrenal Cells: Relevance to the Regulation of Steroid Synthesis’ GHANIM ALMAHBOBI, LINDY J. WILLIAMS, AND PETER F. HALLS Department
of Endocrinology,
Prince
of Wales
Hospital,
The rate of steroid synthesis is regulated by the rate of transport of cholesterol from lipid droplets to mitochondria. We have previously demonstrated that lipid droplets in adrenal cells are tightly attached to intermediate filaments. Here we now show that mitochondria colocalize with intermediate filaments in modified double indirect immunofluorescence and by electron microscopy of extracted adrenal cells. Direct contact between mitochondria and intermediate filaments was established by examination of stereo pairs of electron micrographs from extracted cells. The attachment of both droplets and mitochondria to intermediate filaments suggests possible mechanisms for this form of intracellular transport of cholesterol to mitochondria and hence for the regulation of steroid synthesis. o issz ACTdemic
Press,
Inc.
INTRODUCTION
It has been known for some time that the cholesterol used by steroidogenic cells for the synthesis of steroid hormones is stored in the cytoplasm-much of it in the conspicuous lipid droplets that form a distinctive feature of these cells [l-5]. On the other hand the conversion of cholesterol to steroid hormones begins with a reaction catalyzed by a cytochrome P450 in the inner mitochondrial membrane (P45Oscc) [6]. This enzymatic reaction, called side-chain cleavage of cholesterol (see), results in the formation of the key intermediate pregnenolone. Clearly cholesterol must move from cytoplasm to mitochondria to enter the steroidogenic pathway. It turns out that this process of intracellular transport of cholesterol is the rate-limiting step in the pathway and is specifically stimulated by the trophic hormones ACTH and LH. This step requires the cytoskeleton and, in particular, actin [7-91. In investigating the transport of cholesterol in adrenal cells we found that lipid droplets containing cholesterol ester are attached to intermediate filaments and i Supported * To whom
by NH&MRC reprint requests
Grant 900706. should be addressed.
Randwick,
2031 New
South
Wales,
Australia
remain associated with these filaments after severe extraction with Triton X-100 [4, 51. The contents of the droplets appear to be protected by a capsule containing vimentin [4, 51. To pursue these investigations further we attempted to obtain a reproducible preparation of cytoskeleton in which both lipid droplets and mitochondria are present with the cytoskeleton so that the movement of droplets to mitochondria can be investigated in vitro. For these studies we used both Y-l adrenal tumor cells and primary cultures of bovine fasciculata cells. MATERIALS
AND METHODS
Cell preparation and culture. Primary cultures of bovine fasciculata cells were prepared as described elsewhere [5, lo]. Y-l mouse adrenal tumor cells (American Type Culture) were grown in culture as described elsewhere [4,5,7]. Both cell types were plated at a density of 1 X lOa cells per milliliter in Ham’s F12 plus Dulbecco’s MEM with 12.5% (v/v) horse serum and 2.5% (v/v) fetal calf serum. Production of steroids (20adihydroprogesterone for Y-l cells and cortisol for beef cells) was measured by radioimmunoassays [7, 111 on samples of incubation medium. Cells were grown on glass coverslips, slide chambers, or plastic dishes for morphological studies and in plastic dishes for biochemical assays. Where indicated, experiments were performed in the above medium without serum (serum-free medium). Rupture and extraction of cells. (i) Cells were broken by osmotic lysis using the method of Beckers andcollaborators [12]. (ii) Homogenization was carried out by scraping cells from plastic dishes into a glass-glass Dounce homogenizer in buffer. Cells were ruptured by 20 strokes. (iii) Mild extraction was performed by standing cells in 0.5% Triton X-100 for 5 min as previously described for preparation of cytoskeleton [4, 131. Severe extraction used 1% Triton for 10 minthis procedure was performed once or twice on the same cells as described with individual experiments. To prepare intermediate filaments 1% Triton for 10 min once only was followed by ammonium sulfate [13] in 1% Triton for a further 10 min. Fluorescence microscopy. Y-l and bovine fasciculata cells were grown on glass coverslips and were incubated with serum-free medium containing Rhodamine 123 (10 pglml) for 30 min at 37°C before examination by fluorescence microscopy [14]. In some studies preparations of intermediate filaments were treated with Nile red to stain lipid droplets [4]. Zmmunofluorescence microscopy. This procedure was performed on cells grown on glass slide chambers. The cells were extracted by the mild procedure, by severe extraction, by severe extraction twice, and by treatment with ammonium sulfate to prepare intermediate filaments. (a) Single indirect immunofluorescence was performed to display intermediate filaments or mitochondria exactly as described previously [4].
361
0014-4827t92
Copyright All
rights
0
1992 of reproduction
by Academic in eny
form
$5.00
Press, Inc. reserved.
362
ALMAHBOBI,
WILLIAMS,
AND HALL
MITOCHONDRIA
AND
INTERMEDIATE
FILAMENTS
363
FIG. 1. Fluorescence microscopy of living adrenal cells. Cultured cells were incubated with rhodamine 123 to stain mitochond ria (10 ng ml-‘) for 30 min. (A) Y-l cells and (B) bovine fasciculata. n, nucleus; bar, 2 pm. FIG. 2. Double indirect immunofluorescence microscopy of mildly extracted adrenal cells. Cells were subjected to mild extras cti4 on (see Materials al nd Methods). (A and B) Y-l cells and (C! and D) bovine fasciculata cells. Each pair of panels (A and B; C and D) shows th e same cells at two (excitation wavelengths. Cells were stained for both mitochondria (A and C) and intermediate filaments (B and D). n, nucll E2lE S;bar, 5 w-n. FIG. 3. Double indirect immunofluorescence of the cytoskeleton of Y-l cells. Mitochondria and intermediate filaments were smtai ned by the 43 proc’ edure described under Materials and Methods so that both mitochondria and intermediate filaments can be seen at one f?XC :itation wave length I. n, nucleus; bar, 2.5 pm. FIG. 4. Double indirect immunofluorescence of intermediate filaments. Intermediate filaments prepared from Y-l cells were sts lint sd with anti-cytochl rome oxidase (A), anti-vimentin (B), or anti-vimentin and Nile red (C). Arrows, lipid droplets; n, nucleus; bar, (A) and(B) 51 m, (Cl 2 pm.
364
FIG. 5. Electron thin sections. Arrows tus; m, mitochondrion;
ALMAHBOBI,
microscopy of bovine indicate intermediate bar, 0.125 pm.
fasciculata filaments
WILLIAMS,
AND
HALL
cells. Cells were subjected to lysis (A) or homogenization (B) followed in direct contact with mitochondria. er, dilated endoplasmic reticulum;
(b) Double indirect immunofluorescence microscopy used the standard procedure with four different antibodies raised in four species. The four antibodies were applied separately in the following order: The first antibody for mitochondria was rabbit anti-cytochrome oxidase (kindly provided by Dr. E. Racker, Department of Biochemistry, Cornell University, Ithaca, NY) diluted 1:30. Incubation was for 60 min at room temperature. The second antibody was donkey anti-rabbit IgG conjugated to Texas red diluted 1:40 for 60 min. Intermediate filaments were stained with goat anti-vimentin diluted 1:20 for 60 min and was followed by swine anti-goat IgG conjugated to FITC diluted 1:20 for 30 min. (c) The modified procedure (4:3 immunofluorescence, i.e., four anti-
by preparation of g, Golgi appara-
bodies raised in three species) has been reported in detail [23]. The first three antibodies are the same as those in (b) and the fourth antibody is rabbit anti-goat conjugated to FITC diluted 1:40 for 30 min. In this procedure we substitute rabbit anti-goat IgG conjugated to FITC for the corresponding swine IgG with the result that the new antibody (rabbit anti-goat IgG) will recognize goat anti-vimentin and be recognized by donkey anti-rabbit IgG. Since the latter antibody is indirectly attached to mitochondria (cytochrome oxidase), these structures will be stained with both Texas red and FITC. As a result it is possible to examine both mitochondria and intermediate filaments in the same image (in this case with the blue filter) without the need to change filters and compare two images. At the same time one of
MITOCHONDRIA
AND
INTERMEDIATE
365
FILAMENTS
F ‘IG. 6. Stereo pairs of electronmicrographs of bovine fasciculata cells were prepared. The preparations and thil n sections of the monolayer inte !rrnt diate filaments in direct contact with mitochondria (m). Bar,
cells subjected to lysis. Bovine fasciculata cells were subjected were tilted at -12’ (A) and +12’ (B) and photographed. Arror 0.09 em.
these structures, namely mitochondria, could be identified alone by using the green filter. Other details have been described elsewhere [23]. Electron microscopy. Cells subjected to lysis were processed as monolayers. Homogenized and extracted cells were handled as pellets after scraping from dishes. Samples were fixed in a one-half concentration of Karnovsky fixative for 60 min as described previously [3,5]. Specimens were embedded in Spurr’s resin. Some experiments were performed by the method of Franke et al. using osmium tetroxideglutaraldehyde fixation [15]. Thin sections were examined under a Hitachi 7000 electron microscope. Stereo pairs were prepared by tilting the microscope stage. Analytical procedures. Protein was measured by the method of Bradford [16]. Cytochrome-c oxidase was measured by a published spectrophotometric method [17]. This method is based upon the rate of oxidation of ferrocytochrome c by following the decrease in absorbance at 550 nm. Side-chain cleavage activity was measured as the conversion of cholesterol to pregnenolone. Extracted cells are incubated at 37°C with 10 nM cholesterol, 3.5 mg/ml adrenodoxin reductase, 2 mg/ml adrenodoxin in 50 mM phosphate buffer, pH 7.4. The reaction is started by the addition of 8 mg/ml NADPH. The electron carriers adrenodoxin and its reductase pass electrons from NADPH to the P450 side-chain cleavage enzyme to permit the enzyme to convert cholesterol to pregnenolone (side-chain cleavage of cholesterol). Cholesterol was measured by radioimmunoassay as described by
Mrotek and Hall [7]. The minimal amount of pregnenolone by this method is 0.01 pmol/min/mg protein. Materials. Sources of all materials used were the same described previously [4, 51 except where indicated above.
lysis
detected as those
RESULTS
Fluorescence Microscopy
of Living
Adrenal
Cells
Figure 1 shows fluorescence microscopy of Y-l and bovine fasciculata cells in culture using rhodamine 123 to stain mitochondria in living cells. In both cell types mitochondria appear as filamentous structures but those from bovine fasciculata are considerably more elongated than those from Y-l cells. These organelles are concentrated in the perinuclear region of both cell types.
Immunofluorescence (1) Cytoskeleton. extracted
with
Triton
of Extracted Adrenal X-100
Adrenal Cells
cells of both types were (0.5% v/v for 5 min) and
366
ALMAHBOBI,
WILLIAMS,
AND HALL
FIG. 7. Stereo pairs of electronmicrographs of Y-l cells. Cells were extracted with Triton X-100 using mild conditions (see Materials and Method Is). Thin sections of the cells were tilted to -6” (Al and +6’ (B) and photographed. Arrows indicate intermediate filaments in direct contact with mitochondria (m). n, nucleus; bar, 0.08 pm.
subjected to indirect double immunofluorescence staining using rabbit anti-cytochrome oxidase and donkey anti-rabbit IgG conjugated to Texas red, followed by goat anti-vimentin and swine anti-goat IgG conjugated to FITC, i.e., four antibodies raised in four species (Figs. 2A-2D). It is clear that mitochondria are retained in the cell during mild extraction since intense staining of cytochrome oxidase is seen in punctate structures, especially in the perinuclear region (Figs. 2A and 2C). Staining appears to be confined to mitochondria. Intermediate filaments are clearly revealed by the antibody conjugated to FITC (Figs. 2B and 2D). In order to examine the relative distributions of mitochondria and intermediate filaments we repeated this procedure in a modified form, which we refer to as 4~3 immunofluorescence, as described under Materials and Methods. The intense yellowish green color of the mitochondria seen with the blue filter is quite distinct from the green color of the filaments (Fig. 3). Mitochondria are also distinguishable from intermediate filaments by
morphology. With the green filter only mitochondria appear red as shown in Figs. 2A and 2C (not shown). It is clear that mitochondria are closely associated with and occur along the course of the vimentin filaments. Similar findings were made with bovine fasciculata cells (not shown). (II) Intermediate filaments. Y-l cells were extracted and treated with ammonium sulfate to prepare intermediate filaments (see Materials and Methods) and the filaments were subjected to indirect double immunofluorescence staining as in Fig. 2 (four antibodies raised in four species). Mitochondria are not visible as such but patches of red staining can be seen. The procedure used has destroyed mitochondria but these residual fragments of stained material can be seen (Fig. 4A). Intermediate filaments are clearly outlined as before with the aid of anti-vimentin and second antibody (Fig. 4B). Similar findings are seen with bovine fasciculata cells (not shown). Finally by using anti-vimentin with a second antibody and the lipid stain Nile red, we see that
MITOCHONDRIA
AND
INTERMEDIATE
FIG. 8. Stereo pairs of bovine fasciculata cells mildly extracted with Triton. were prepared. Sections were tilted to -6” (A) and +6’ (B) and photographed. mitochondria (m). n, nucleus; bar, 0.07 pm.
in the intermediate filament preparation, lipid droplets are to be seen in keeping with our earlier reports [4, 51 (Fig. 4C). (III) Severe extraction. When cells of either type are subjected twice to severe extraction (see Materials and Methods) mitochondria are entirely removed from the preparation. Evidently the removal of mitochondria is not specifically related to the use of ammonium sulfate (data not shown). Electron
Microscopy
Bovine fasciculata cells were broken by two procedures designed to produce as little disruption of cellular architecture as possible, namely osmotic lysis and gentle homogenization. A thin section of a cultured bovine fasciculata cell subjected to lysis is shown in Fig. 5A. In addition to the usual cellular structures, lo-nm filaments are seen in contact with mitochondria. A similar association is seen in thin sections of homogenized Y-l cells (Fig. 5B). In order to examine contact points between filaments
FILAMENTS
367
Cells were extracted by the mild procedure and thin sections Arrows indicate intermediate filaments in direct contact with
and mitochondria, stereo pairs were made by tilting specimens. Figure 6 shows a thin section of a bovine fasciculata cell subjected to lysis. Intermediate filaments are clearly revealed and well-preserved mitochondria are seen with filaments passing between two of these organelles. Figures 7 and 8 show stereo pairs of thin sections of cytoskeletons from Y-l and bovine fasciculata cells, respectively. Careful examination of the stereo pairs shows that direct contact occurs between intermediate filaments and mitochondria in cells of both types. Furthermore preparation of intermediate filaments, which involves an ammonium sulfate treatment, removes all mitochondria (not shown). These findings are consistent with those described above for immunofluorescence microscopy. Characterization
of Mitochondria
Table 1 shows that the preparation of cytoskeleton (mild extraction) leaves significant amounts of cytochrome oxidase activity in the extracted cells-both Y-l and fasciculata. The need for Triton to reveal maxi-
368
ALMAHBOBI,
TABLE Cytochrome
WILLIAMS,
1
Oxidase
in Adrenal
Cells
Activity (nmol/min/lO’
cells)
Treatment
Cell Y-l Bovine fasciculata
Homogenate
Homogenate and Triton x-100
Mild extraction
19
102
49
18
65
43
Intermediate filaments 12 0.8
Note. Adrenal cells were homogenized and aliquots were taken for measurement of cytochrome oxidase. The homogenate was then treated with Triton X-100 (v/v) and aliquots were again taken for assay of cytochrome oxidase activity. Other cells were subjected to mild extraction or used to prepare intermediate filaments. Cytochrome oxidase activity was measured on the cell residues. These data were a result of two separate experiments and the figures are rounded to the closest values.
ma1 activity in the homogenate makes it difficult to establish an exact recovery of cytochrome oxidase. However, approximately half of this enzyme activity remains after mild extraction. The intermediate filaments have lost most of the cytochrome oxidase. The small amount seen in the filament preparations is consistent with faint mitochondrial staining seen in the immunofluorescence studies shown in Fig. 4A. In other experiments we found that the mitochondria of mildly extracted cells are still capable of catalyzing side-chain cleavage of cholesterol, albeit at a greatly reduced rate compared to that of mitochondria prepared from homogenized cells (1.5 and 0.2 pmol of pregnenolone/min/mg protein in Y-l and bovine fasciculata cells, respectively). These values are significantly greater than background (0.01 pmol/min/mg protein). The mitochondria of the extracted cells require addition of electron carriers to catalyze side-chain cleavage since these proteins are readily removed from mitochondria. DISCUSSION
The two important findings to emerge from these studies are first that we can prepare an adrenal cell extract from which cytosol, plasma membrane, and much of the internal structure of the cell have been removed, leaving no more than cytoskeleton, lipid droplets, and mitochondria, and second the demonstration that in such extracted adrenal cells functional mitochondria are attached to intermediate filaments. Since the ratelimiting step in steroid synthesis-one that is stimulated by ACTH and its second messenger cyclic AMPinvolves the transport of cholesterol from lipid droplets
AND HALL
to mitochondria, it would be helpful to use a preparation of cells that contains these two structures. Such apreparation should be permeable to large molecules and it would be desirable to remove most of the remaining cell structure. With such a preparation, extracts, molecules, and organelles could be added back to restore the process of intracellular transport of cholesterol. It is possible to measure transport of cholesterol to mitochondria and the effect of ACTH on this process in intact adrenal cells [7-91. Experimental approaches to transport of cholesterol would be facilitated by using the simplest subcellular system that contains the two structures involved, namely lipid droplets and mitochondria. In searching for such a system we discovered that lipid droplets containing cholesterol ester are very tightly attached to intermediate filaments [4,5]. We present here a mild extraction procedure for adrenal cells that leaves all three structures, i.e., droplets, filaments, and mitochondria, in the extracted cell. The conditions used produce sufficient clearing of cell contents to permit thorough morphological studies with light and electron microscopy. Moreover the mitochondria are capable of performing the side-chain cleavage of cholesterol. In addition the extracted cell contains approximately onehalf of the original cellular cytochrome oxidase. The evidence for attachment of mitochondria to intermediate filaments includes localization in fluorescence microscopy and direct visualization of contact points on electron microscopy. Although the resolution of fluorescence microscopy does not allow visualization of contact points, the distribution of individual mitochondria with individual filaments seen in our preparations indicates colocalization of these two structures. In view of the repeated washing of cells involved in fluores-
cence microscopy the mitochondrial membrane must be attached to some internal structure. Moreover when we compare intact living cells with mildly extracted cells we see no change in the distribution of mitochondria. Although the retention of mitochondria following mild extraction does not in itself indicate binding of these structures to intermediate filaments, all the above considerations together with the evidence for direct contact seen on electron microscopy make it clear that mitochondria are firmly bound to intermediate filaments. Since more than 50% of a mitochondrial marker enzyme is retained on mild extraction, it. is very unlikely that the mitochondria are removed from their normal intracellular location to become artifictitiously attached to the intermediate filaments. Moreover during mild extraction some mitochondrial fragments remain clearly visible in fluorescence microscopy, indicating tight binding of these organelles to intermediate filaments. It would appear that some form of attachment must be necessary to hold the fragments within the disrupted cell. Finally the scattered patches of mitochondrial staining seen
MITOCHONDRIA
AND INTERMEDIATE
after severe extraction suggest the possibility that fragments of mitochondria remain bound to intermediate filaments. These filaments are virtually the only structures present after severe extraction which removes actin and tubulin from the cell [4]. Mitochondria remaining after mild extraction are morphologically and biochemically damaged, appearing as small vesicular structures which are however still capable of catalyzing the side-chain of cholesterol. It is also of interest to note that the two cell types show certain differences on extraction. For example, the fasciculata cells appear to suffer greater morphological damage for the same intensity of extraction. It is however useful to be able to prepare extracted cells from both a cell line and primary cultures of normal cells since both Y-l [ 181 and bovine fasciculata cells have been widely used in studies of adrenal cell function and because of important differences between cytoskeletons of normal and transformed cells. Attachment of mitochondria to intermediate filaments has been reported for other cell types [19, 201. In skin fibroblasts a protein from intermediate filaments called lEF24 is found to be closely associated with mitochondria [ 191. Clearly it will be important to determine the mechanism of attachment of these two structures in adrenal cells. In the meantime it is important to note that the association of mitochondria with intermediate filaments reported here is more than a surface contact between these structures. The components of inner membrane (side-chain cleavage enzyme and cytochrome oxidase) are attached to the filaments, indicating an intimate binding between intramitochondrial enzymes and intermediate filaments. The intermediate filaments seen in Figs. 2B and 2D show a punctate appearance that we interpret as further evidence of the intimate association between mitochondrial contents and intermediate filaments. This striped appearance (which is seen only where mitochondria are present) probably represents a change in the organization of vimentin where the filaments are in contact with mitochondria. However, we cannot exclude some leakage of fluorescent signal in the FITC channel. The observation that both functional mitochondria and cholesterol-containing lipid droplets are attached to intermediate filaments, together with earlier studies by other workers showing lipid droplets in intimate contact with mitochondria (so-called docking) [21], suggests possible mechanisms whereby the vectorial characteristics of intermediate filaments could facilitate targeting of droplets to mitochondria. Previous studies from this laboratory showed that microfilaments are involved in the increase in the transport of cholesterol to mitochondria in response to ACTH and cyclic AMP [ 7Received Revised
October 3,199l version received
February
12, 1992
369
FILAMENTS
91. This may point to a contractile mechanism which could act in concert with the directional properties of intermediate filaments to facilitate transport of droplets to mitochondria or vice versa. The idea is made attractive by recent studies showing that in permeabilized fibroblasts ATP promotes reorganization of intermediate filaments as the result of contraction of peripheral actomyosin [22]. The authors are grateful to Dr. Adrian Zammit for the assays of cytochrome oxidase and side-chain cleavage and to Eve Smith for typing the manuscript. We are also grateful to the staff of the Electron Microscopy Unit at the University of New South Wales and to the Director, Dr. M. R. Dixon.
REFERENCES 1. 2.
Rhodin, J. A. G. (1971) J. UZtrastruct. Res. 34, 23-71. Almahbobi, G., Silberzahn, N., Fakhri, R., and Silberzahn, (1985) Arch. Anat. Microsc. Morphol. Exp. 74, 193-203.
3.
Almahbobi, G., Papadopoulos, V., Carreau, P. (1988) Biol. Reprod. 38,653-665.
4. 5.
Almahbobi, Almahbobi,
6.
Hall, Ed.),
G., and Hall, G., Williams,
P.
S., and Silberzahn,
P. F. (1990) J. Cell Sci. 97, 679-687. J. L., and Hall, P. F. (1992) J. Cell Sci.
101,383-393. P. F. (1987) in Hormonal Proteins and Peptides (Li, C. H., Vol. XIII, pp. 89-125, Academic Press, New York. J. J., and Hall,
Mrotek,
Hall, P. F., Charponnier, C., Nakamura, (1979) J. Biol. Chem. 254,9080-9084.
M.,
9.
Osawa, 1342.
J. Cell Biol.
S., Betz,
P. F. (1977)
G., and Hall,
Biochemistry
16,3177-3181.
I. a.
P. F. (1984)
10.
Gospodarowicz, D., Ill, C. R., Hornsby, (1977) Endocrinology 100, 1080-1089.
11.
Yanagibashi, K., Ohno, Y., Kawamura, (1988) Endocrinology 123, 2075-2082. Beckers, C. J. M., Keller, D. S., and Balch, 523-534.
12. 13.
Fey, E. G., Wan, 1973-1984.
14.
Johnson, L. V., Walsh, M. L., and Chen, Acad. Sci. USA 77,990-994.
15.
Franke, Keenan, Bradford,
16.
K. M.,
and Penman,
and Gabbiani, 99,1335-
P. J., and M.,
and
Gill,
G. N.
Hall,
P. F.
W. E. (1987)
S. (1984)
G.
Cell 50,
J. Cell Biol.
L. B. (1980)
W. W., Ludez, M. R., Kartenbeck, J., Zerban, T. W. (1976) J. Cell Biol. 69,173-195. M. (1976) Anal. Biochem. 72, 248-254.
98,
Proc. Natl. H., and
17.
Wharton, D. C., and Tzagoloff, A. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., Eds.), Vol. 10, pp. 245-250, Academic Press, San Diego.
18.
Kowal, J. (1970) Recent Prog. Horm. Res. 26,623-677. Mose-Larsen, P., Bravo, R., Fey, S. J., Small, J. V., and Celis, J. E. (1982) Cell 31,681-692. Lin, A., Krockinglic, G., and Penman, S. (1990) Proc. Natl. Acad. Sci. USA 87,8565-8569.
19. 20.
119, 611-618.
21.
Merry,
22.
Tint, I. S., Hollenbeck, P. J., Verkhovsky, A. B., Surgucheva, J. F., and Bershadsky, A. D. (1991) J. Cell Sci. 98, 375-384. Almahbobi, G., and Hall, P. F. (1992) Hi&o&em. J., in press.
23.
B. J. (1975)
J. Anat.
