Cell Motility and the Cytoskeleton 17:15&166 (1990)
Dynamic Properties of Intermediate Filaments: Disassembly and Reassembly During Mitosis in Baby Hamster Kidney Cells Ellen Rae Rosevear, Manette McReynolds, and Robert D. Goldman Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, Chicago A morphological analysis of the organizational changes in the type I11 intermediate filament (IF) system in dividing baby hamster kidney (BHK-21) cells was carried out by immunof luorescence and immunoelectron microscopy. The most dramatic change occurred during prometaphase, when the typical network of long 10-nm-diameterIF characteristic of interphase cells disassembled into aggregates containing short 4-6 nm filaments. During anaphase-telophase, arrays of short IF reappeared throughout the cytoplasm, and, in cytokinesis, the majority of IF were longer and concentrated in a juxtanuclear cap. These results demonstrate that the relatively stable IF cytoskeletal system of interphase cells is partitioned into daughter cells during mitosis by a process of disassembly and reassembly. This latter process occurs in a series of morphologically distinct steps at different stages of the mitotic process. Key words: cytoskeletal dynamics, IF depolymerization, type 111 IF regulation
INTRODUCTION Of the three major cytoskeletal systems of mammalian cells, two have been shown to undergo predictable changes in their supramolecular organization and distribution during mitosis. Cytoplasmic microtubule complexes depolymerize as cells enter mitosis and subsequently repolymerize to form the mitotic apparatus [Brinkley et al., 19801. Microfilament bundles disassemble in late prophase and later form the contractile ring of microfilaments at the cleavage furrow during cytokinesis [Schroeder, 19761. However, much less is known about the fate of intermediate filaments (IF) during mitosis. In the few keratin (IF types I and 11)- and keratin/ vimentin (IF type 111)-containing cells that have been studied to date, some 10-nm-diameter IF appear to disassemble into aggregates of their constituent proteins during mitosis, whereas IF in other cell types form a cage or aggregate of 10 nm filaments around the mitotic apparatus [Blose, 1979; Zieve et al., 1980; Horwitz et al., 1981; Franke et al., 1982; Celis et al., 1983; Lane et al., 1982; Brown et al., 1983; Jones et al., 1985; Kitajima et al., 19851. There are discrepancies regarding the content 0 1990 Wiley-Liss, Inc.
of the aggregates found in mitotic cells. For example, Franke et al. [I9821 observed that the aggregates in mitotic HeLa cells contained both keratin and vimentin. On the other hand, Jones et al. [I9851 observed that the aggregates in mitotic HeLa cells contained keratin, whereas the vimentin system remained filamentous. Therefore, the details of the organization of IF in mitotic cells remain unclear. The literature on the fate of vimentin- or vimentin/ desmin-containing IF in fibroblast-like cell lines during mitosis is confusing. In some cases it has been reported that a cage of IF is maintained around the mitotic apparatus [Hynes and Destree, 1978; Gordon et al., 1978; Zieve et al., 1980; Aubin et al., 1980; Blose and Bushnell, 1982; Lane et al., 19821. However, Starger, et al.  reported that IF are rarely found in BHK-21 cells
Received December 27, 1989; accepted July 10, 1990 Address reprint requests to Robert D. Goldman, Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 6061 I .
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in metaphase. More recently, we have shown that IF proteins of prometaphase-metaphase BHK-2 1 cells appear to be localized primarily in randomly distributed, nonfilamentous aggregates by immunofluorescence [Chou et al., 19891. It is quite possible that some of these contradictions in the literature are the result of observations of different stages (e.g., metaphase vs. anaphase) of the mitotic process in different cell lines. Based on these considerations, we have initiated studies aimed at clarifying the organizational changes in IF networks in a single cell line, BHK-21, throughout the entire process of cell division. Our results demonstrate that the cytoplasmic IF system undergoes significant changes in its organization and states of polymerization at each of the major stages of mitosis. MATERIALS AND METHODS Cell Cultures and Drug Treatment
colchicine for 15, 30,45, and 60 min producing enriched populations of cells in prometaphaseimetaphase, metaphaseianaphase, ana- phaseitelophase, and telophaseicytokinesis at the various time points, respectively. Cells in these cultures are referred to as colchicine-reversed cells. Anti bodies
A rabbit polyclonal antiserum that reacts with both BHK vimentin and desmin (IF antibody) [Yang et al., 19851 and a mouse monoclonal antibody directed against P-tubulin (tubulin antibody) (Amersham, Arlington Heights, IL) were used as primary antibodies in these studies. The secondary antibodies used were fluoresceinconjugated goat antirabbit IgG (Southern Biotech. Assoc., Inc., Birmingham, AL) and rhodamine-conjugated goat antimouse IgG (Kirkegaard & Perry Laboratories, Inc., Gaitherburg, MD). For immunoelectron microscopic studies, we used either 5 or 10 nm colloidal goldlabeled goat antirabbit IgG (Janssen Life Sciences Products, Beerse, Belgium).
Stock cultures of BHK-21 cells were grown in 100 mm plastic petri dishes in Dulbecco’s modified Eagle’s medium as described by Yang et al. . For light microscopic studies of mitotic cells, subconfluent Fluorescence Microscopy Single and double label indirect immunofluorescultures were trypsinized (0.5% trypsin, 0.5 mM EDTA, phosphate-buffered saline without added Ca2+ and cence was carried out on normal and colchicine-reversed Mg2+ [PBSa: 0.171 M NaCl, 0.003M KCl, 0.006M cultures as described by Jones et al. . In some Na+-K+ phosphate, pH 7.41) and replated at a density preparations, cells were slightly flattened prior to fixaof lo5 cellsiml culture medium onto poly-L-lysine- tion for immunofluorescence procedures using the agacoated coverslips for approximately 18 hr. This method rose overlay method of Fukui et al. [1986, 19871. Norprovides a maximum number of mitotic cells for light mal unflattened cells appeared to be identical in their overall morphology to slightly flattened cells, especially and electron microscopic studies. Another method of obtaining mitotically enriched with regard to immunofluorescence patterns. However, cultures took advantage of the minimal adhesive proper- the details of immunofluorescence patterns were freties of rounded up dividing cells. Populations of mitotic quently easier to distinguish in the slightly flattened cells were prepared from logarithmic growth phase cul- cells. This was due to the reduction of the relatively tures by mechanically shaking off loosely attached, di- higher background fluorescence levels derived from out viding cells grown in flasks [Dickerman and Goldman, of focus images in the thicker, unflattened cells (data not 19731. The suspended cells were collected by centrifu- shown). gation at 800-1000 rpm in an IEC HN-SII centrifuge and Some coverslips were counterstained with 0.1% then resuspended in culture medium. Two milliliter ali- (wt ./vol.) solution of 4’-6’diamidino-2-phenyl-dihydroquots were plated onto poly-L-lysine-coated coverslips. chloride (DAPI) in McIlraines buffer (0.2 M Na2HP0,, The cells were allowed to attach to the coverslips for 30 pH 7.0, 0.1 M citric acid) for 1 min in a moist chamber. min at 4°C. When these coverslips were returned to This permitted the visualization of chromosomes 37”C, the cells progressed through mitosis normally. [Williamson and Fennel, 1975; Lin et al., 19771. CovThis method yielded cell populations that were approxi- erslips were mounted in PBSa onto slides by sealing with fingernail polish. PBSa was used as a low-refractivemately 95% mitotic. For some light microscopic and all electron micro- index mounting medium for observing the same cell with scopic studies, actively growing cultures were treated for phase-contrast and fluorescence optics. Light micro3-4 hr with 1-1.5 kg colchicine (Sigma Chemical Co., scopic observations were made with a Zeiss photomicroSt. Louis, M0)iml of culture medium to obtain mitoti- scope I11 (Carl Zeiss, Inc., Thornwood, NY) equipped cally arrested cells. These arrested cells were released with a I11 RS epifluorescence system and phase-contrast from mitotic inhibition by removing the colchicine-con- optics. Micrographs were taken on Kodak Plus-X film taining medium and replacing it with normal medium. (Eastman Kodak, Inc., Rochester, NY) and developed in These cultures were allowed to recover in the absence of Diafine (Acufine, Inc., Chicago, IL).
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Procedures for conventional transmission electron microscopic observations of flat embedded BHK-2 1 cells were described in Starger et al. . For some observations, cells were fixed and lysed simultaneously in a solution containing 0.1% glutaraldehyde, 0.1% Triton X- 100 in MES (2-[N-morpholino]-ethanesulfonic acid [Sigma Chemical Co., St. Louis, MO]) buffer (0.1 M MES [pH 6.61, 0.5 mM MgSO,, 2 mM EGTA). Electron microscopic observations were made on a Jeol 1200EX electron microscope at an accelerating voltage of 60 kV (Jeol USA, Peabody, MA). Immunoelectron microscopic observations using the IF antibody were carried out using the protocol described by Yang et al. . Cells on coverslips were fixed and lysed simultaneously in 0.1% glutaraldehyde, 0.1% Triton X-100 in MES buffer for 30-45 sec. After three rinses in MES buffer, the cultures were further extracted with 0.15% Triton X-100 in MES buffer for 3 min. After three more rinses, the free aldehyde groups were reduced with 0.5 mg/ml NaBH, in MES buffer for 20 min, and the fixed cells were preabsorbed with normal goat serum diluted 1:lO in MES buffer (20 min, room temperature). The coverslips were then rinsed in 0.1% bovine serum albumin (BSA; Sigma Chemical Co.) in MES buffer for 5 min, and overlaid with primary antibody for 60 min at room temperature in a moist chamber. They were then washed three times in 0.1% BSA in MES buffer. The coverslips were exposed to 5 or 10 nm collojdal gold-conjugated goat antirabbit IgG (Janssen Life Science Products) in 0.1% BSA in MES buffer for 60 min at room temperature, followed by three washes in this buffer. The resulting preparations were further fixed in 1% glutaraldehyde and processed for electron microscopy as described above. Similarly fixed and lysed cells were processed for immunofluorescence as a control. Rhodamine-conjugated goat antirabbit IgG (Southern Biotech. Assoc., Inc., Birmingham, AL) was used as the second antibody in these preparations.
the perinuclear region towards the cell surface [ Starger and Goldman, 1977; Goldman et al., 19861 (Fig. 1). This interphase IF network is altered as cells enter prophase, as indicated by the initiation of chromosome condensation, nuclear envelope breakdown, and cell rounding (Fig. 2a,c). In this stage, the IF staining pattern becomes heavily concentrated in the juxtanuclear region containing the paired centrioles or centrosomes, which are revealed by tubulin antibody staining (Fig. 2b,d). Prometaphase cells are distinguished by early stages in the formation of the mitotic apparatus and the increased condensation of chromosomes (Fig. 3a,b). At this stage, the IF antibody reveals many fluorescent spots varying in size and shape, surrounded by a diffusely staining background (Fig. 3c). As the metaphase spindle forms, most of the diffuse background fluorescence disappears, and larger and more intensely staining spots are seen throughout the cytoplasm with the IF antibody (Fig. 3f,i). During anaphase and telophase, the IF antibody reveals a more fibrillar pattern throughout the cytoplasm. At this stage, the region of highest fluorescence intensity is usually at the poles of the mitotic spindle (Fig. 4a-c). This general organization of IF is maintained through late cytokinesis (Fig. 4d-f), the major significant change being a progressive increase in the intensity of IF fluorescence at the poles (Fig. 4c,f,i). When cells have reentered interphase and are in mid- to late G1, daughter cells are recognized by their similar shapes and close proximity. In these cells, prominent juxtanuclear IF caps are seen in the same region as the microtubule-organizing center (centrosomal region; Fig. 4g-i). These IF caps are very similar to those seen in trypsinized and replated cells during the early stages of spreading [see Wang and Goldman, 19781. Electron Microscopic Observations
During the entire interphase period, an extensive IF network is maintained throughout all regions of the cytoplasm between the nuclear and cell surfaces (Fig. 5). This corresponds to the immunofluorescence pattern seen in Figure 1. Late prophase/early prometaphase is RESULTS distinguished by condensing chromosomes and the initial Light Microscopic Observations stages of nuclear envelope breakdown (Fig. 6a,b). Large At the light microscopic level of resolution, numbers of IF are seen in regions adjacent to the disinchanges in the distribution of IF during mitosis were tegrating nuclear envelope (Fig. 6b). These ultrastrucmonitored by indirect irnmunofluorescence with the IF tural observations correspond to the fluorescence obserantibody (see Materials and Methods). To determine mi- vations shown in Figure 2. It has been extremely difficult to locate prototic stages, the mitotic apparatus was visualized by staining with tubulin antibody [Brinkley et al., 1976, metaphaselmetaphase cells in electron microscopic prep19801, DAPI staining to visualize chromosomes arations of normal cultures. Therefore, cultures treated [Williamson and Fennel, 1975; Lin et al., 19771, and with colchicine for 3-4 hr contain a high percentage of rnitotically arrested cells. These can be removed by mephase-contrast microscopy. During interphase, IF in BHK-2 1 cells extend from chanical displacement. The resulting cell suspension is
Dynamic Properties of Filaments
Fig. I . a: Fluorescence micrograph of a typical interphase BHK-21 cell stained with the IF antibody preparation. b: Phase-contrast micrograph of same cell. Note the fluorescent fibrillar pattern which extends from the nucleus towards the cell surface. Bar = 10 p,m.
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Fig. 2. a: Phase-contrast micrograph of a cell in late prophase. Fluorescence micrographs of the same cell: stained with the monoclonal tubulin antibody, which reveals the paired centrioles (b), stained with DAPI (c), stained with the polyclonal IF antibody (d). Note that IF are concentrated in the centrosomal regions. Bar = 10 p,m.
collected by centrifugation and cultured in the absence of colchicine for various time intervals (see Materials and Methods). These colchicine-reversed cells are able to proceed through the completion of mitosis as determined by light microscopy (Fig. 7). This protocol provides a great enrichment for cells in prometaphaselmetaphase at 15 min following reversal. The majority of these cells contain fluorescent spots as revealed by IF antibody staining (Fig. 7a-c) and thus represent ideal preparations for electron microscopic observations. In contrast to the late prophase/early prometaphase cells (Fig. 6), no obvious 10-nm-diameter IF can be found in thin sections of prometaphase or metaphase cells fixed for electron microscopy 15 min after the removal of colchicine (Fig. 8). As a further control, we examined several metaphase cells not treated with colchicine and the ultrastructural observations are identical (not shown). The IF protein-enriched spots seen by fluorescence microscopy are extremely difficult to recognize in conventional thin sections studied by electron microscopy. However, we have been able to distinguish numerous fibrillar regions of medium electron density distributed in
the cytoplasm of prometaphase and metaphase cells (Fig. 8a,b). The frequency and distribution of these regions becomes more apparent in lysed-fixed cell preparations (Fig. 9a) (see Materials and Methods). We believe that these regions represent the bright fluorescent spots revealed by the anti-IF antibody (see Figs. 3c,f,i and 7c). Furthermore, we have observed that cells prepared with this electron microscope procedure exhibit similar patterns of spots (as seen, for example, in Fig. 3f), when they are observed by indirect immunofluorescence (data not shown). The ultrastructure of the aggregates seen in the lysed-fixed cell preparations consists primarily of 4-6nm-diameter filamentous material (Fig. 9b). To establish more convincingly that these regions are equivalent to the fluorescent spots, the immunogold-labeling method has been employed. Typical results are depicted in Figure 10, which demonstrates that the majority of gold particles are found in association with the fibrillar aggregates. The gold particles are located primarily at the periphery of these aggregates, most likely due to the preembedment gold-labeling technique employed in these preparations (see Materials and Methods). Under
Dynamic Properties of Filaments
Fig. 3 . Micrographs of a prometaphase cell stained with the monoclonal tubulin antibody using the agar overlay method (a), DAPI (b), and the IF polyclonal antibody (c). Note that in c the IF pattern is finely speckled, and these speckles are surrounded by diffuse fluorescence. (d-f) Micrographs of a polar view of a metaphase BHK-21 cell stained as above: tubulin antibody (d), DAPI (e), and IF antibody (0.
Note that in f the IF antibody stains larger spots, and there is less diffuse background fluorescence. Same type of preparation as above (metaphaselearly anaphase cell), but a longitudinal view of the mitotic apparatus. g: Tubulin antibody. h: DAPI. i: IF antibody. Bar = 10 )*m.
these conditions, it seems likely that antibody molecules would be unable to penetrate the fixed IF protein aggregates but would bind to surface exposed antigenic sites. Control preparations in which only the second goldtagged antibody is used show no labeling. Thin sections of anaphase and telophase cells show a significant increase in the number of 10-nm-diameter IF and no obvious 4-6 nm fibrillar aggregates (Fig.
1 la,b). IF are seen primarily in loose aggregates concentrated at the spindle pole regions but can also be found elsewhere in the cytoplasm. During cytokinesis and entry into G1, while the midbody still remains visible, numerous long 10-nm-diameter IF are visible by conventional thin section electron microscopy, especially in the perinuclear region (Fig. 12). These ultrastructural observations parallel the changes seen in IF organization from
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Fig. 4. Fluorescence micrographs of a cell in anaphase viewed with tubulin antibody (a), DAPI (b), IF antibody (c). Cell in late telophase-cytokinesis stained with tubulin antibody (d), DAPI (e), IF antibody (0. Freshly divided daughter cells stained with tubulin antibody (g), DAPI (h), IF antibody (i). All three cells prepared using the agarose overlay method. Bar = 10 p m .
Dynamic Properties of Filaments
Fig. 5 . Electron micrograph of a spread interphase cell showing large numbers of long IF (arrows), which are seen throughout the cytoplasm. Bar = 0.25 IJ-m.
anaphase through cytokinesis by fluorescence micros-
diameter IF are present continuously through the cell cycle in fibroblast-like cell lines, forming a cage around COPY. the spindle during mitosis [Gordon et al., 1978; Hynes and Destree, 1978; Zieve et al., 1980; Horwitz et al., DISCUSSION 1981; Blose and Bushnell, 1982; Lane et al., 19821. The IF system of BHK-21 cells is significantly al- However, other studies of BHK-21 cells [Blose and tered during mitosis. Furthermore, this altered IF protein Bushnell, 1982; Lane et al., 19821 have not taken into is localized in protofilamentous aggregates during account changes in IF organization as a function of the prometaphase and metaphase. These aggregates do not precise stage of the mitotic process. Therefore, these contain 10-nm-diameter IF, but do contain 4-6 nm di- latter results may be explained by the rapid transitions in ameter short filaments. This latter form of IF protein IF structure that take place from one stage of mitosis to disappears as short 10-nm-diameter IF reappear in the next. In other words, the time frame during which IF anaphase and telophase. Subsequently, the long IF, are obviously disassembled is very short relative to the which typify interphase IF networks, are formed prima- entire mitotic cycle, and this period could easily be overrily, but not exclusively, in the centrosomal regions of looked. As was indicated in the Introduction, the formation the two daughter cells during and following cytokinesis. These observations show that the IF of BHK-21 cells of aggregates during mitosis is not unique to BHK-21 undergo a cyclic change in their organizational state dur- cells. Similar observations of disassembled IF proteins have been reported in several cell lines containing one or ing mitosis. In contrast to our results, other laboratories have more of types I, 11, and I11 IF systems [Lane et al., 1982; reported that significant numbers of polymerized 10-nm- Horwitz et al., 1981; Franke et al., 1982; Brown et al.,
Fig. 6. Electron micrographs of a thin section of a late prophaseiearly prometaphase cell. a: Low magnification showing the state of condensation of the chromosomes and the beginnings of nuclear envelope breakdown. b: Higher magnification view of the area marked by the rectangle in a. IF (arrow) are seen in the juxtanuclear region. Bar in a = 1 k m . Bar in b = 0.2 p m .
Dynamic Properties of Filaments
Fig. 7 . Triple label fluorescence observations of a late prometaphasei metaphase cell maintained in the absence of colchicine for 15 minutes and stained with the tubulin monoclonal antibody (a), DAPI (b), and the rabbit IF antibody (c). Triple label fluorescence micrographs of an anaphase cell fixed 45 min following colchicine removal. Tubulin Triple label monoclonal antibody (d), DAPI (e), and IF antibody (0.
fluorescence observations of a cell in cytokinesis 60 min after colchicine removal. g: Monoclonal tubulin antibody. h: DAPI. i: IF antibody. Note that there are no significant differences between the cells following colchicine treatment and the normal, untreated cells seen in Figures 3 and 4. Bar = 10 p m .
1983; Celis et al., 1983; Jones et al., 1985; Kitajima et 19891. Chou et a].  have characterized two endogal., 19851. Unfortunately, these latter studies have not enous IF protein kinase activities in mitotic BHK-21 included detailed light and electron microscopic analyses cells. In vitro phosphorylation of vimentin and desmin of each mitotic stage to show the disassembly and reas- catalyzed by one of these endogenous kinases is coincisembly process. Based on these studies and our results, dent with the disassembly of 10-nm-diameter IF and the it appears that the formation of the protofilamentous ag- formation of 4-6-nm-diameter filaments as seen by neggregates in prometaphase and metaphase of BHK-2 1 ative stain electron microscopy. We believe that these cells helps to establish an equal distribution of IF protein latter phosphorylated filaments are analogous to those to daughter cells [Franke et al., 1982; Jones et al., 19851. found in the 4-6 nm filamentous aggregates seen in It is also possible that the IF disassembly process con- prometaphase-metaphase cells. tributes to the changes in cell shape during the rounding Other laboratories have noted similar increases in the phosphorylation of IF protein isolated from mitotic up of fibroblasts in mitosis. Recently, it has been shown that the morphological cells and the disassembly of IF phosphorylated in vitro rearrangements of the BHK IF system are coincident with A and C kinases [Evans and Fink, 1982; Celis et a]., with significant increases in the phosphorylation levels 1983; Fey et al., 1983; Evans, 1984, 1989; Inagaki et of vimentin and desmin during mitotis [Chou et a]., al., 1987, 1988; Tolle et al., 1987; Inagaki et al., 19881.
Fig. 8. Electron micrographs of a metaphase cell fixed in glutaraldehyde without detergent lysis 15 min after colchicine reversal (see Materials and Methods). a: Several regions of medium electron density represent potential IF protein aggregates (arrows). Microtubules are present indicating recovery from the colchicine treatment (mt). b: High-magnification view of the area in the rectangle seen in a. A fibrillar substructure is observed in area delineated by arrows. Bar in a = I +m. Bar in b = 0.2 pn.
Fig. 9. Electron micrographs of a fixed-lysed metaphase cell (see Materials and Methods). a: Numerous IF protein aggregates are seen throughout the cytoplasm (arrows). Microtubules are present indicating recovery from colchicine treatment (mt). b: Higher magnification of the IF protein aggregate seen in the rectangle in a. The aggregate consists primarily of 4-6 nm filaments. Bar in a = 1 pn. Bar in b = 0.2 IJ.m.
Fig. 10. a: Immunogold labeling of IF protein aggregates in a prometaphaselmetaphase BHK-21 cell with the primary IF protein antibody and the secondary goat antirabbit IgG conjugated with 5 nm colloidal gold (arrows). b: High-magnification view of two fibrillar regions surrounded by gold particles. Note the close association of the gold to the filamentous aggregates. The gold is located at the periphery
of the aggregate probably due to the preembedment labeling technique used (see Material and Methods). In these preparations, the fibrils of the aggregates appear to be approximately twice as thick as those depicted in Figure 9b. This is most likely due to the coating of the bound antibody. Bar in a = I pm. Bar in b = 0.2 km.
Dynamic Properties of Filaments
Fig. 11. a: Electron micrograph of an anaphase cell. Note microtubules (MT). b: High-magnification view of the area outlined by the rectangle shows short IF (arrows) in the spindle pole region of the cell. Bar in a = 1 p m . Bar in b = 0.25 F m .
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Fig. 12. Electron micrograph of a cell in late cytokinesis 60 min following colchicine reversal. a: The cells are joined by a midbody (arrow). b: Higher-magnification view shows the extensive juxtanuclear IF network in the rectangular region indicated in a (arrow). Bar in a = 1 Km. Bar in b = 0.2 Km.
Dynamic Properties of Filaments
Dickerman, L . , and Goldman, R.D. (1973): A rapid method for production of binucleate cells. Exp. Cell Res. 83:433-436. Evans, R.M. (1984): Peptide mapping of phosphorylated vimentinEvidence for a site-specific alteration in mitotic cells. J. Biol. Chem. 29:5372-5375. Evans, R.M. (1989): Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities. J. Cell Biol. 108:6778. Evans, R.M., and Fink, L.M. (1982): An alteration in phosphorylation of vimentin type intermediate filaments is associated with mitosis in cultured mammalian cells. Cell 29:43-52. Fey, S.J., Larsen, P.M., and Celis, J.E. (1983): Evidence for coordinated phosphorylation of keratins and vimentin during mitosis in transformed human amnion cells: Phosphate turnover of modified proteins. FEBS Lett. 157:165-169. Franke, W.W., Schmid, E., Grund, C . , and Geiger, B. (1982): Intermediate filament proteins in nonfilamentous structures: Transient disintegration and inclusion of subunit proteins in granular aggregates. Cell 30:103-113. Fukui, Y., Yumura, S . , and Yumura, T.K. (1987): Agar-overlay imACKNOWLEDGMENTS munofluorescence: High resolution studies of cytoskeletal components and their changes during chemotaxis. In Prescott, This work has been supported by grants from the D. (eds.): “Methods in Cell Biology, Vol. 28.” New York: National Cancer Institute and the National Institute for Academic Press, pp. 347-356. General Medical Sciences. We thank all members of our Fukui, Y., Yumura, S . , Yumura, T.K., and Mori, H. (1986): Agar laboratory for their advice and encouragement during overlay method: High-resolution lmmunofluorescence for the these studies and Ms. Laura Davis for helping in the study of contractile apparatus. Methods Enzymol. 134:573580. preparation of the manuscript. Goldman, R.D., Goldman, A.E., Green, K.J., Jones, J.C.R., Jones, S . , and Yang, H.-Y. (1986): Intermediate filament networks: Organization and possible functions of a diverse group of cyREFERENCES toskeletal elements. J. Cell Sci. Suppl. 5:69-97. Aubin, J.E., Osborn, M . , Franke, W., and Weber, K . (1980): Inter- Gordon, W.E., Bushnell, A , , and Burridge, K . (1978): Characterization of the intermediate (10-nm)filaments of cultured cells mediate filaments of vimentin-type and cytokeratin type are using an autoimmune rabbit antisera. Cell 13:249-261. distributed differently during mitosis. Exp. Cell Res. 124:93Honvitz, B., Kupfer, H . , Eshar, Z., and Geiger, B. (1981): Reorga109. nization of arrays of prekeratin filaments during mitosis. Exp. Blose, S.H. (1979): Ten-nanometer filaments and mitosis: MainteCell Res. 134:281-290. nance of structural continuity in dividing endothelial cells. Hynes, R.O., and Destree, A.T. (1978): 10 nm filaments in normal Proc. Natl. Acad. Sci. USA 76:3372-3376. and transformed cells. Cell 13:151-163. Blose, S.H., and Bushnell, A. (1982): Observations on vimentin 10nm filaments during mitosis in BHK-21 cells. Exp. Cell Res. Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K . , Nishi, Y., and Sato, C. (1988): Intermediate filament reconstitution in 142:57-62. vitro-The role of phosphorylation on the assemblyBrinkley, B.R., Fistel, S.M., Marcum, J.M., and Pardue, R.L. disassembly of desmin. J . Biol. Chem. 263:5970-5978. (1980): Microtubules in cultured cells; indirect immunofluorescent staining with tubulin antibody. Int. Rev. Cytol. 63: Inagaki, M . , Nishi, Y., Nishizawa, K., Matsuyama, M . , and Sato, C. (1987): Site-specific phosphorylation induces disassembly of 59-95. vimentin filaments in vitro. Nature 328:649-652. Brinkley, B.R., Fuller, G . M . , and Highfield, D.P. (1976): Tubulin antibodies as probes for microtubules in dividing and non-di- Jones, J.C.R., Goldman, A.E., Yang, H.-Y., and Goldman, R.D. (1985): The organizational fate of intermediate filament netviding mammalian cells. In Goldman, R.D., Pollard, R., and works in two epithelial cell types during mitosis. J . Cell Biol. Rosenbaum, J. (eds.): “Cell Motility, Book A,” Cold Spring 100:93-102. Harbor, NY: Cold Spring Harbor Publications, pp. 435-456. Brown, D.T., Anderton, B.H., and Wylie, C.C. (1983): Alterations in Kitajima, Y., h o e , S . , Yoneda, K . , Mori, S . , and Yaoita, H. (1985): Alterations in the arrangement of keratin-type intermediate filthe organization of cytokeratins in normal and malignant huaments during mitosis in cultured human keratinocytes. Eur. J. man colonic epithelial cells during mitosis. Cell Tissue Res. Cell Biol. 38:219-225. 233:6 19-628. Celis, J.E., Larsen, P.M., Fey, S.J., and Celis, A. (1983): Phosphor- Lane, E.B., Goodman, S.L., and Trejdosiewicz, L.K. (1982): Disruption of the keratin filament network during epithelial cell ylation of keratin and vimentin polypeptides in normal and division. EMBO J. 1:1365-1372. transformed mitotic human epithelial amnion cells: Behavior of keratin and vimentin filaments during mitosis. J. Cell Biol. Lin, M.S., Comings, D.E., and Alfi, 0 , s . (1977): Optical studies of interaction of 4’-6-diamidino-2-phenylindole with DNA and 97:1429-1434. metaphase chromosomes. Chromosoma 60: 15 -25, Chou, Y.-H., Rosevear, E . , and Goldman, R.D. (1989): Phosphorylation and disassembly of intermediate filaments in mitotic Schroeder, T.E. (1976): Actin in dividing cells: Evidence for its role in cleavage but not mitosis. In Goldman, R.D., Pollard, T . , cells. Proc. Natl. Acad. Sci. USA 86:1885-1889.
However. these studies have not been correlated with changes in morphology of IF in situ. Taken together, these data suggest that the regulation of the degree of phosphorylation and the sites of phosphorylation and dephosphorylation of IF proteins all appear to play important roles in regulating the state of IF assembly in vivo. Further studies on the properties of the endogenous kinases in BHK-21 cells are now in progress [Chou et al., 19891 and should shed additional light on the exact molecular mechanisms regulating the IF system during mitosis. It should be emphasized, however, that phosphorylation and subsequent dephosphorylation may represent only two of numerous factors yet to be described that are necessary for regulating the remodeling of the IF system during mitosis.
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