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Am J Physiol Cell Physiol 279: C369–C374, 2000. Clathrin in mitotic spindles CURTIS T. OKAMOTO, JEANA MCKINNEY, AND YOUNG Y. JENG Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089-9121 Received 10 August 1999; accepted in final form 23 February 2000 conventional and “muscle” isoforms of clathrin heavy chain have been reported in various tissues (3). Together, these data suggest that clathrin may possess diverse functions in a multicellular organism. Here we show by immunofluorescence with two anti-clathrin heavy chain monoclonal antibodies (MAbs) and anticlathrin light chain polyclonal antibodies that clathrin is localized to the mitotic spindle of mammalian cells. Clathrin heavy chain was also detected on Western blots of isolated spindles. These results suggest either a role for clathrin in mitosis or a cell cycle-dependent regulatory mechanism for clathrin localization. EXPERIMENTAL PROCEDURES the role of clathrin in the regulation of receptor-mediated endocytosis from the plasma membrane and of budding of transport vesicles from the trans-Golgi network have been made at the morphological, biochemical, cellular, genetic, and atomic structural levels (3, 16, 23, 25, 29, 32). Clathrin is comprised of two subunits, a heavy chain of relative molecular mass (Mr) of 170 kDa and a light chain of Mr of 32 kDa. In mammalian cells, several isoforms of the light chains have been identified. Three heterodimers assemble into three-legged structures known as triskelions that, in turn, polymerize to form cages on budding membranes in vivo and in vitro. Recently, clathrin has been localized to other intracellular membranes, such as endosomes, and may function to regulate trafficking at these sites as well (6, 8, 19, 28). Moreover, a second isoform of the clathrin heavy chain has been cloned that is apparently expressed ubiquitously in mammalian fetal tissues, but is predominantly expressed in skeletal muscle in the adult (12). Alternative mRNA transcripts of both the Cells and antibodies. Madin-Darby canine kidney (MDCK) cells (strain II) of European Molecular Biology Laboratory parentage were obtained from Dr. Keith Mostov (Univ. of California, San Francisco). CV-1 cells were obtained from Dr. Sarah Hamm-Alvarez (Univ. of Southern California). The MAb X-22 hybridoma (2) was purchased from the American Type Tissue Collection (Washington, DC), and the hybridoma supernatants were used neat for immunostaining. Anticlathrin heavy chain MAb 23 and anti-␥-adaptin MAb were purchased from Transduction Laboratories (Lexington, KY). The rabbit polyclonal antiserum against the conserved region of mammalian light chains was a kind gift from Dr. Frances Brodsky (Univ. of California, San Francisco). Goat anti-mouse IgG conjugated to indocarbocyanine (Cy3) or FITC and goat anti-rabbit IgG conjugated to FITC secondary antibodies were purchased from Jackson Immunological Research Laboratories (Bar Harbor, ME). ProLong antifade mounting medium and propidium iodide were purchased from Molecular Probes (Eugene, OR) and used according to the manufacturer’s instructions. BSA, fish skin gelatin, anti-␣-tubulin MAb, nocodazole, and brefeldin A (BFA) were purchased from Sigma Chemical (St. Louis, MO). Goat anti-mouse IgG secondary antibody coupled to horseradish peroxidase was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence detection kit for horseradish peroxidase was purchased from Pierce Chemical (Rockford, IL). Molecular mass markers for SDS-PAGE were purchased from Bio-Rad Laboratories (Hercules, CA). Immunofluorescence. Cells were plated at subconfluent densities onto glass coverslips. Cells were fixed either in 3.7% formaldehyde, diluted from a 37% stock solution (Fisher Scientific, Pittsburgh, PA) in PBS for 20 min at room temperature, or in cold (⫺20°C) methanol. Formaldehyde-fixed glands were permeabilized by 0.5% Triton X-100 in PBS for Address for reprint requests and other correspondence: C. T. Okamoto, Dept. of Pharmaceutical Sciences, School of Pharmacy, Univ. of Southern California, Los Angeles, CA 90089-9121 (E-mail: [email protected]). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Madin-Darby canine kidney cells; CV-1 cells; brefeldin A SIGNIFICANT ADVANCEMENTS IN DEFINING http://www.ajpcell.org 0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society C369 Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017 Okamoto, Curtis T., Jeana McKinney, and Young Y. Jeng. Clathrin in mitotic spindles. Am J Physiol Cell Physiol 279: C369–C374, 2000.— Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anticlathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells. C370 CLATHRIN IN MITOTIC SPINDLES RESULTS Two MAbs against clathrin heavy chain, MAb X-22 (2) and MAb 23, and a rabbit polyclonal antiserum against clathrin light chain were used to stain two Fig. 1. Indirect immunofluorescent labeling of mitotic spindles in Madin-Darby canine kidney (MDCK) and CV-1 cells by anti-clathrin heavy chain monoclonal antibody (MAb) X-22 and MAb 23 and anticlathrin light chain polyclonal antiserum. a and b: subconfluent MDCK cells doublestained with propidium iodide for nuclear and chromosomal staining (a) and MAb X-22 for anti-clathrin heavy chain (b). c-e: subconfluent MDCK cells stained with MAb X-22. f: subconfluent MDCK cells stained by anti-clathrin heavy chain MAb 23. g: CV-1 cells stained with MAb 23. h: subconfluent MDCK cells stained with the rabbit anti-light chain polyclonal antiserum. Bar ⫽ 5 m. types of cultured cells, MDCK cells and CV-1 cells, by indirect immunofluorescence. MAb 23 recognizes the NH2 terminus of clathrin heavy chain (the epitope is within a fragment spanning amino acids 4–171), and MAb X-22 binds nearer to the COOH terminus (17). The polyclonal antiserum recognizes a consensus sequence found in all mammalian clathrin light chains (amino acids 23–40) (1). As shown in Fig. 1, immunofluorescent staining by all antibodies results in the typical, well-documented punctate intracellular staining pattern in interphase MDCK and CV-1 cells (Fig. 1, b-d and Fig. 2a). In addition, perinuclear staining is usually prominent; this staining pattern is thought to represent staining of the trans-Golgi network. In mitotic cells, punctate intracellular staining was also visible. However, unexpectedly, under the conditions employed here, mitotic spindles in both cell types were also strongly immunoreactive with all three anti-clathrin antibodies: MAb X-22 (Fig. 1, b-e), MAb 23 (Fig. 1, f and g), and polyclonal anti-light chain (Fig. 1h). In mitotic cells, the anti-clathrin immunoreactivity in mitotic spindles was significantly stronger than that observed on intracellular membranes. The pattern of anti-clathrin staining of mitotic spindles is clearly distinct from that of chromosomal staining (Fig. 1, a and b), but is reminiscent of staining of the microtubule array in mitotic spindles (26). Anti-clathrin staining was evident from an early stage of mitosis (late prophase, Fig. 1d) to later stages (telophase, Fig. 1e). Thus, MAb X-22 and MAb 23, MAbs that bind to distinct and widely separated Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017 20 min at room temperature. Nonspecific binding sites were blocked by incubation with 3% BSA and 0.66% fish skin gelatin in PBS. Cells were incubated with primary antibody, followed by fluorescently labeled secondary antibody, and mounted in ProLong antifade mounting medium. Cells were observed and photographed on a Zeiss Axioskop epifluorescence microscope or images were collected using a Nikon PCM Quantitative Measuring High-Performance Confocal System attached to a Nikon TE300 Quantum upright microscope. Parenthetically, the manner in which the cells were processed for immunofluorescence was important for detecting anti-clathrin immunoreactivity in the mitotic spindle, particularly for MAb X-22. Anti-clathrin immunoreactivity in mitotic spindles was not observed when cells were fixed and permeabilized with cold methanol, a popular method of fixing and permeabilizing cells for staining with MAb X-22 (data not shown). For BFA experiments, cells were incubated with BFA (20 g/ml) for 20 min at 37°C and subsequently processed for immunofluorescence. Thymidine-nocodazole blocking of cultured cells to enrich for mitotic cells was performed according to Zieve et al. (34). Isolation of mitotic spindles and crude clathrin-coated vesicles from brain. Spindles were isolated from mitotic CV-1 cells according to a protocol adapted from Zieve et al. (34). Crude clathrin-coated vesicles were isolated according to the protocol of Pearse and Robinson (20). SDS-PAGE was run according to the protocol of Laemmli (11), and Western blots were performed according to the protocol of Towbin et al. (30). CLATHRIN IN MITOTIC SPINDLES C371 epitopes on clathrin heavy chain, and the anti-clathrin light chain antiserum all immunolabel the mitotic spindle. These results suggest that clathrin is present in mitotic spindles. To confirm that clathrin is localized to the mitotic spindle, subconfluent MDCK cells were double-labeled by immunofluorescence for clathrin light chain and tubulin (Fig. 2, a and b). In mitotic cells, there is substantial colocalization of clathrin light chain and tubulin in the spindle. An additional experiment was performed in which mitotic cells were enriched by arresting cells with a thymidine-nocodazole block and subsequently releasing them from the block (34). Shown in the confocal microscopic images in Fig. 2, c and d, are subconfluent MDCK cells after being released from a thymidine-nocodazole block and stained for clathrin heavy chain. Anti-clathrin staining is present in spindle-like staining patterns, and even appears to stain spindles in cells with hemispindles and multiple spindles. These results confirm the im- munofluorescent localization of clathrin to the mitotic spindle. For some populations of clathrin-coated membranes and vesicles, the association of clathrin and clathrin adaptor proteins with their target membranes is regulated by the small GTPase ADP ribosylation factor (ARF). BFA reversibly inhibits the function of ARF by inhibition of GDP:GTP exchange factors for ARF (4), and incubation of cells with BFA prevents the recruitment of ARF-dependent vesicular coat proteins, such as the Golgi-associated coatomer protein complex (COPI) and the clathrin adaptor proteins AP-1 and AP-3, onto target membranes (7, 24, 31). The sensitivity of clathrin in mitotic spindles to BFA was assessed in CV-1 cells that were treated with BFA and subsequently double-labeled for ␥-adaptin and clathrin light chain (Fig. 3). Addition of BFA to CV-1 cells results in a characteristic dispersion in Golgi-associated anti-␥adaptin staining in interphase cells (Fig. 3, a and c). However, BFA did not affect the localization of clathrin Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017 Fig. 2. a and b: confocal micrographs of double immunofluorescent labeling of clathrin light chain (a) and ␣-tubulin (b) in subconfluent MDCK cells. Clathrin light chain and microtubules in mitotic spindles are colocalized. c and d: confocal micrographs of clusters of mitotic MDCK cells labeled with anti-clathrin heavy chain MAb X-22. Clusters of mitotic cells were produced by releasing cells from a thymidine-nocodazole block as described in EXPERIMENTAL PROCEDURES. Spindle staining is apparent in cells with hemi-, single, and multiple spindles. Interphase cells are barely visible in the background. All bars ⫽ 5 m. C372 CLATHRIN IN MITOTIC SPINDLES light chain to the mitotic spindle (Fig. 3, b and d). Anti-clathrin heavy chain MAb X-22 was also used to stain cells treated with BFA. In the presence of BFA, Golgi-associated staining was lost in interphase cells, whereas staining of the spindle in mitotic cells remained intact (data not shown). Thus the association of clathrin with the mitotic spindle appears to be mechanistically distinct from its association with Golgi membranes and may be independent of ARF. Alternatively, if ARF is involved in the binding of clathrin to the mitotic spindle, ARF may be regulated by a BFAinsensitive exchange factor (4). Given the apparently significant amount of clathrin immunoreactivity associated with the mitotic spindle, one would predict to find anti-clathrin immunoreactivity associated with isolated mitotic spindles. Spindles were isolated from mitotic CV-1 cells and were analyzed on Coomassie blue-stained gels and by Western blot (Fig. 4). The profile of proteins associated with the isolated spindles as analyzed by SDS-PAGE appears to be similar to previously published results (Fig. 4A, lane 1) (34). Anti-tubulin immunoreactivity is shown in Fig. 4B, lanes 1 and 2, to confirm the presence and enrichment of tubulin, a major component of isolated spindles. Although proteins are visible in the region where clathrin heavy chain is expected to migrate (Mr of ⬃170 kDa), based upon the lack of a distinctly visible clathrin heavy chain band, clathrin heavy chain does not appear to be a significant component of isolated mitotic spindles (Fig. 4A, lane 1). A sample of crude-coated vesicles from hog brain, in which clathrin heavy chain is a major component, is shown for comparison (Fig. 4A, lane 2). However, by immunoblot analysis of isolated mitotic spindles (Fig. 4C, lanes 1 and 2), clathrin is indeed detectable, although not apparently enriched, in isolated spindles. In mitotic cells, the strong immunolabeling of mitotic spindles with anti-clathrin antibodies suggests that the spindle may represent a major clathrin-containing organelle. However, the association of clathrin with the spindle may be quite labile, particularly under the conditions used to isolate spindles, because clathrin does not appear to be enriched in isolated mitotic spindles. Moreover, at this time, we cannot rule out the possibility that clathrin in this spindle preparation may represent clathrin that is Fig. 4. A: Coomassie blue-stained SDS-gel of isolated mitotic spindles from CV-1 cells (20 g; lane 1) and crude-coated vesicles from brain (20 g; lane 2). B: Western blot of total cell lysate (20 g; lane 1) and isolated mitotic spindles (20 g; lane 2) from CV-1 cells probed with anti-tubulin MAb. C: Western blot of total cell lysate (20 g; lane 1) and isolated mitotic spindles (20 g; lane 2) from CV-1 cells probed with anti-clathrin heavy chain MAb 23. The positions of molecular mass standards (in kDa) are shown. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017 Fig. 3. Association of clathrin with the mitotic spindle is not affected by brefeldin A (BFA). Untreated (a and b) or BFA-treated (c and d) CV-1 cells were fixed and double-labeled for ␥-adaptin (a and c) and clathrin light chain (b and d) and viewed by confocal microscopy. The perinuclear Golgi-associated localization of ␥-adaptin (arrows) is characteristically affected by BFA. The localization of clathrin light chain to the mitotic spindle is not affected by BFA (arrowheads). The exposures for the images of clathrin light chain have been adjusted to visualize spindle staining. Such images are indicators of the relative intensities of staining between interphase and mitotic cells. Bar ⫽ 10 m. CLATHRIN IN MITOTIC SPINDLES associated with the detergent-insoluble cytoskeleton of mitotic cells; in interphase cells, clathrin has been shown to be associated with the detergent-insoluble cytoskeleton (9). DISCUSSION vitro assay for the role of clathrin in the mitotic spindle would greatly benefit from the ability to isolate spindles with clathrin bound to them. However, the biochemical data presented here suggest that the association of clathrin with the mitotic spindle may be somewhat labile, given the inability to copurify significant amounts of clathrin with isolated spindles. This lability may be the reason that clathrin appears to be a minor component of isolated spindles and, therefore, has not previously been reported to be a component of isolated spindles. A third possibility is that the anti-clathrin antibodies may be recognizing a closely related isoform of clathrin with a novel function in the mitotic spindle. Both of the anti-clathrin heavy chain MAbs bind to regions of clathrin heavy chain that are highly conserved between the conventional and “muscle” isoform of clathrin (3) and, therefore, could be recognizing an isoform of clathrin with a distinct function in the mitotic spindle. However, one argument against the possibility that spindle clathrin is the muscle isoform is that clathrin light chain is present in the spindle. The muscle isoform of clathrin has amino acid substitutions in the light chain binding region that make binding to light chain highly unfavorable (32); thus the presence of light chain in the spindle would not be expected. The final possibility is that localization of clathrin to the mitotic spindle might represent a novel, cell cycledependent, regulatory mechanism for the subcellular localization of clathrin. The robust staining of clathrin in the mitotic spindle suggests that the spindle may contain a significant fraction of the total cellular clathrin in mitotic cells. One characteristic of mitotic cells is that clathrin-mediated endocytosis is inhibited (21, 22). Although clathrin-coated pits are still present in mitotic cells, early stages of clathrin-coated pits (shallow domes and wide necks) are abundant relative to clathrin-coated pits in the later stages (i.e., narrow necks) of formation (21). A massive recruitment of clathrin to the mitotic spindle would, therefore, deplete the cellular pool available for the completion of coated pits and vesicles. This mechanism, working in conjunction with that of the inhibition of the interaction of other components of coated pits at the plasma membrane, such as ␣-adaptin, Eps 15, and epsin, due to the phosphorylation of Eps 15 and epsin during mitosis (5), would reinforce a block in endocytosis. Alternatively, clathrin may be recruited to the spindle during mitosis to ensure a roughly equal division of clathrin between the two daughter cells, similar to the process for the mitotic division of the Golgi apparatus (13). In summary, all of these possibilities that may account for clathrin localization to the mitotic spindle are highly speculative, and further investigation is warranted. The identification of clathrin in the mitotic spindle suggests that clathrin may be a protein with multiple functions or a protein with a localization that is subject to cell cycle-dependent regulation. Its further characterization with respect to its role in the mitotic spindle should lead to additional insight into clathrin function. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017 The data presented here suggest that clathrin is present in mitotic spindles. Interestingly, we are not the first to report the presence of clathrin in the mitotic spindle. Maro et al. (14), using their own anti-clathrin polyclonal antibodies, observed immunofluorescently labeled clathrin in the second metaphase spindle of an unfertilized mouse egg and in mitotic spindles of cells in early embryos. Thus we have confirmed and extended their seminal observations. The data from these reports together suggest that clathrin localization to the mitotic spindle may be a fundamental property of dividing cells. To date, the only other report of clathrin’s involvement in mitosis was its role in the regulation of cytokinesis in Dictyostelium, where it was found that a strain of Dictyostelium that lacks clathrin was defective in cytokinesis (18). The role of clathrin in mitotic spindles needs to be defined at the morphological, cellular, and biochemical levels. One possibility that may account for the distinctive and characteristic anti-clathrin staining of mitotic spindles is that the anti-clathrin antibodies are staining clathrin-coated vesicles or membrane tubules that are tethered to microtubules in the mitotic spindle. These vesicles may be remnants of fragmented Golgi (26) or other clathrin-coated organelles, analogous to mitotic fragments of Golgi membranes coated with the COPI coatomer complex (15). However, the pattern of anti-clathrin staining of the spindle is distinct from that of anti-Golgi staining associated with the mitotic spindle: the anti-Golgi staining is clearly found in “clumps” that are clustered around the spindle poles (24, 26). The absence of an effect of BFA on anticlathrin staining of the mitotic spindle (Fig. 2) also argues against, but does not completely rule out, this first possibility, because the association of clathrin and, more directly, the AP-1 clathrin adaptor with Golgi membranes is regulated by ARF (27, 33). In fact, Robinson and Kreis (24) have shown that in mitotic cells treated with BFA, the staining for COPI coatomer and the AP-1 clathrin adaptor becomes diffuse, similarly to that observed for interphase cells. A second possibility for the presence of clathrin immunoreactivity in mitotic spindles is that clathrin is serving a novel scaffolding or mechanochemical function in the spindle. Such a function for clathrin might be inferred from the distinct staining of muscle cell sarcomeres by MAb X-22 (10). Clathrin may be involved in the assembly and/or maintenance of ordered cellular structures such as the sarcomere and the mitotic spindle. If clathrin serves such a function, one prediction would be that the mitotic spindles in clathrin-minus Dictyostelium may be abnormal; unfortunately, the morphology of mitotic spindles in this strain of Dictyostelium was not investigated (18). An in C373 C374 CLATHRIN IN MITOTIC SPINDLES We thank Dr. Sarah Hamm-Alvarez for helpful suggestions, we acknowledge continual help from and generosity of Drs. Frances Brodsky and Shu-Hui Liu from the Brodsky lab, and we thank Dr. Francesca Santini for drawing our attention to the Maro paper. We acknowledge the Confocal Microscopy Subcore for the Univ. of Southern California Center for Liver Diseases, supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Core Center Grant PO3 DK-48522. This work was supported by a Grant-in-Aid from the National American Heart Association and NIDDK 51588 (C. T. Okamoto). REFERENCES 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 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