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Vol. 18, Issue 12, 4750-4761, December 2007
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*Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, United Kingdom; and Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom
Submitted February 15, 2007;
Revised August 24, 2007;
Accepted September 6, 2007
Monitoring Editor: Erika Holzbaur
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The process of cytokinesis is driven by the formation and constriction of an actomyosin II contractile ring and by targeted membrane addition (Glotzer, 2005). Membrane delivery is first required during cleavage furrow ingression to increase the surface area of the two daughter cells and later during the abscission process to seal the two newly formed cells (Strickland and Burgess, 2004; Albertson et al., 2005). Although membrane vesicles delivered into the cleavage furrow were thought to be derived from the secretory pathway (Skop et al., 2001), endocytosis and membrane recycling have also now been shown to be required for cytokinesis (Gerald et al., 2001; Schweitzer and D'Souza-Schorey, 2002; Fielding et al., 2005; Wilson et al., 2005). Endocytosis resumes during late cytokinesis and endocytic vesicles or vesicles from the recycling compartment are transported into the midbody region of the cleavage furrow (Schweitzer et al., 2005).
Because myosin VI is an actin-based motor protein involved in endocytic and exocytic membrane-trafficking pathways in interphase cells (Buss et al., 2004), we decided to test whether it was involved in cytokinesis in dividing cells. An increasing number of proteins associated with the actin and microtubule cytoskeleton and with membrane-trafficking pathways have been shown to play a role in cytokinesis. In a recent proteomic study by Skop et al. (2004), 160 candidate midbody proteins were identified, including myosin VI and its binding partner GAIP-interacting protein COOH terminus (GIPC). Furthermore, functional studies in Caenorhabditis elegans using RNA interference showed that both proteins display defects in germline cytokinesis (STE/GON) (Skop et al., 2004).
Myosin VI is a retrograde motor because, unlike all the other classes of myosins, it moves toward the minus end of actin filaments (Wells et al., 1999); therefore, it is expected to have unique and distinct intracellular functions. The targeting and functions of myosin VI at different intracellular locations involve a variety of different binding partners such as disabled-2 (Morris et al., 2002), GIPC (Bunn et al., 1999), and optineurin (Sahlender et al., 2005), and they also require binding to phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane (Spudich et al., 2007). GIPC is a postsynaptic density 95/disc-large/zona occludens domain-containing protein that interacts with myosin VI and with the cytoplasmic domains of several transmembrane receptors at the plasma membrane, where it may regulate endocytic receptor trafficking or intracellular signaling (De Vries et al., 1998).
In this study, we examined the role of myosin VI and its binding partner GIPC during cell division in mammalian cells. In prophase in early mitosis, myosin VI is present in the pericentriolar region. At the onset of cytokinesis, myosin VI is recruited to the contractile ring in the cleavage furrow, colocalizing with actin and myosin II; and after ingression, it is found in the region of the midbody, where it colocalizes with GIPC. In time-lapse video microscopy, myosin VI can be seen associated with vesicles transported into and out of the midbody region, suggesting that it is involved in membrane trafficking during cytokinesis. Only the full-length "active" myosin VI but not the "nonfunctional" tail domain is targeted into the cleavage furrow and midbody. Overexpression of this dominant-negative tail inhibits delivery of vesicles containing the transferrin receptor (TfR) into the midbody region, and it results in multinucleated cells. In addition depletion of myosin VI or its binding partner GIPC, by using small interfering RNA (siRNA), leads to defects in cytokinesis. Furthermore, loss of myosin VI in KD cells causes a dramatic delay in progression through metaphase and in chromosome alignment.
These results highlight for the first time a role for myosin VI in the progression of mitotic cells through metaphase and demonstrate that myosin VI plays an important role in membrane trafficking events during cytokinesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), a rabbit antibody to calf thymus nonmuscle myosin II (Drenckhahn et al., 1983), a monoclonal antibody (mAb) to green fluorescent protein (GFP) (Q-biogen, Cambridge, United Kingdom), a polyclonal antibody to GFP (Molecular Probes, Leiden, The Netherlands); a mAb to 
-tubulin (Sigma, Gillingham, United Kingdom), a mAb to transferrin receptor (Zymed, Cambridge BioScience, Cambridge, United Kingdom), and a rabbit polyclonal antibody to GIPC and a rabbit antibody to actin (Sigma). Rhodamine-coupled phalloidin (Sigma) was used to visualize F-actin filaments in immunofluorescence.
Cell Culture and Transfection
HeLa cells were cultured and transfected as described previously (Sahlender et al., 2005). After 16–18 h, the cells were either used for live cell imaging or they were fixed for immunofluorescence. Madin Darby canine kidney (MDCK) II cells were cultured in DMEM containing 10% (vol/vol) fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Myosin VI or the tail domain tagged with GFP at the N terminus was cloned into the 
pMEP4 vector (Warner et al., 2003). Protein expression was induced for 24 h with 100 µM ZnCl2. For selection of stable MDCK cell lines, MDCK cells were transfected as described above and selected with 200 µg/ml hygromycin (Roche Diagnostics, Lewes, United Kingdom). Single clones were isolated, and the population of highly expressing cells enriched by fluorescence-activated cell sorting. Immunoblotting of stable cell lines showed that the concentration of expressed protein was at least equal to that of endogenous myosin VI (Au et al., 2007).
Knockdown of Myosin VI and GIPC by siRNA
siRNA knockdown experiments were performed as described previously (Sahlender et al., 2005). "On target Plus" SMARTpools designed and supplied by Dharmacon (Cramlington, United Kingdom) were used for knock down of myosin VI and GIPC. To perform siRNA-rescue experiments, three silent point mutations were made in the target sequence of one of the myosin VI SMARTpool oligos using QuikChange (Stratagene, Amsterdam, The Netherlands). A stable HeLa cell line was generated expressing GFP-tagged mutated siRNA-resistant myosin VI by using pIRESneo2 (Clontech-Takara BioEurope, Saint-Germain-en-Laye, France).
Indirect Immunofluorescence Microscopy
HeLa and MDCK cells were processed for immunofluorescence microscopy as described previously (Buss et al., 2001). To remove cytosolic proteins before fixation, MDCK cells expressing GFP-myosin VI or GFP-tail were prepermeabilized with 0.05% saponin in cytosol buffer (Morris and Cooper, 2001). Cells were analyzed and photographed using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Welwyn Garden City, United Kingdom).
Fluorescence Live Cell Microscopy
For time-lapse fluorescence microscopy, MDCK cells expressing GFP-myosin VI were grown on 25-mm-diameter glass coverslips. Images were acquired with a 63x objective and a spinning disk confocal head (PerkinElmer-Cetus, Beaconsfield, United Kingdom) coupled to Zeiss Axiovert 135TV fluorescence microscope. Images were captured with ORCA-ER camera (Hamamatsu, Bridgewater, NJ) controlled by the UltraView imaging software (PerkinElmer-Cetus). Fluorescence images were acquired every 10 s and processed using ImageJ software (http://rsb.info.nih.gov/ij/).
Differential Interference Contrast (DIC) Live Cell Microscopy
For time lapse DIC microscopy control or knockdown (KD) HeLa cells were grown on 25-mm round coverslips that were mounted in an Attofluor cell chamber (Molecular Probes), which was filled with CO2-independent medium (Invitrogen, Paisley, United Kingdom) and placed in a heated stage at 37°C on a Zeiss Axiovert 200M microscope (Carl Zeiss). DIC images were acquired every 2 min with Openlab and analyzed using Velocity software (Improvision, Coventry, United Kingdom).
Whole Mount Immunocytochemistry
Whole mount immunocytochemistry was performed as described previously (Stoorvogel et al., 1996; van Dam and Stoorvogel, 2002). HeLa cells were cultured overnight on Formvar carbon-coated gold grids mounted on glass coverslips. The cells were washed with ice-cold phosphate-buffered saline (PBS), and cytosolic proteins were extracted with PBS containing 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mg/ml saponin (Sigma Chemical, Poole, Dorset, United Kingdom) before fixation with 1% paraformaldehyde at 4° for 1 h. The cells were then incubated in blocking buffer (PBS, 0.5 mg/ml saponin, 20 mM glycine, 0.1% cold water fish gelatin, and 0.02% NaN3) before immunolabeling (Slot et al., 1991) with a polyclonal antibody to myosin VI tail (Buss et al., 1998) followed by protein A 10-nm gold.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Buss et al., 2001; Warner et al., 2003). To investigate the localization of myosin VI in dividing cells, we immunostained MDCK cells with different myosin VI tail antibodies and we also examined stable MDCK cell lines expressing GFP-tagged myosin VI. Both approaches showed that at the onset of mitosis, during prophase and prometaphase, myosin VI is present in a vesicular, punctate staining pattern concentrated at the centrosome/spindle poles (Figure 1A, a–c, and B, g–i). In late anaphase/early telophase, myosin VI is recruited to the plasma membrane surrounding the ingressing cleavage furrow (Figures 3A and 4, a–c), and in later stages of cytokinesis, it is concentrated on either side of the midbody region (Figures 1A, d–f, and B, j–l; 2; 3, A–C; and 4, d–f). This very distinct recruitment of myosin VI to the spindle poles and the cleavage furrow and subsequently to the cytoplasmic bridge on either side of the midbody during cytokinesis was observed in immunofluorescence microscopy initially with different affinity-purified antibodies to myosin VI and then confirmed by expression of full-length GFP-tagged myosin VI (Figure 1B, g–l). To obtain higher resolution images of myosin VI in the midbody region, we performed whole mount immunoelectron microscopy on HeLa cells grown on Formvar carbon-coated gold grids. In this method, cytosolic proteins were removed before fixation and labeling with our rabbit polyclonal antibody to myosin VI tail. Consistent with the immunofluorescence data myosin VI is present along cytoskeletal structures on either side of the very electron-dense area of the midbody (Figure 2a). Enlarged images highlight the association of myosin VI with small intracellular vesicular structures of 
50 nm (Figure 2, b–d, arrows) and with fibrous cytoskeletal elements.
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), the no-insert isoform is important for protein sorting in the exocytic pathway, leading to delivery to the basolateral domain in polarized epithelial cells (Au et al., 2007). All three myosin VI isoforms that are expressed in MDCK cells show the same localization in mitotic cells, indicating that in contrast to the localization of myosin VI isoforms in interphase cells, the inserts do not seem to play a role in differential targeting of myosin VI in mitotic cells (data not shown).
Myosin VI Is Present on Vesicular Structures Trafficking Into and Out of the Midbody Region
During cytokinesis, constriction of the cleavage furrow requires an increase in plasma membrane surface area. This is achieved by inserting new membrane into the cleavage furrow, which is either derived from the secretory or endocytic pathways. In interphase cells myosin VI is associated with endocytic and exocytic vesicles and the Golgi complex (Buss et al., 1998; Warner et al., 2003). Therefore, to explore whether myosin VI plays a role in directing membrane vesicles into the cleavage furrow, we used time-lapse video microscopy on MDCK cells stably expressing GFP-myosin VI. The dynamic behavior of myosin VI tagged with GFP during cytokinesis can be seen in still images in Figure 3. At the onset of cytokinesis, myosin VI is recruited and concentrated in the region of the plasma membrane, where the actin/myosin II contractile ring is being assembled (Figure 4) and the plasma membrane is starting to invaginate (Figure 3A and Supplemental Movie 1). During cleavage furrow ingression, myosin VI becomes very concentrated in the narrow cytoplasmic bridge on either side of the midbody. At the end of cytokinesis, myosin VI is gradually lost from the midbody region. In addition to the concentration of myosin VI in the cleavage furrow, we observed the recruitment of myosin VI to the plasma membrane outside the cleavage furrow and to punctate structures in the polar regions surrounding the spindle poles, which are most likely membrane vesicles (Supplemental Movie 1). Myosin VI is present on punctate structures that are moving from both daughter cells into the cytoplasmic bridge as shown on selected frames in Figure 3B (Supplemental Movie 2). In the left daughter cell, a small myosin VI-containing structure (highlighted by a arrow) is budding from a larger fluorescent structure before moving rapidly in 30 s into the midbody region. In this movie, equal numbers of myosin VI-positive spots are moving into and out of the cleavage furrow. This movement of myosin VI-containing punctate structures is not only observed in the cleavage furrow but also a large number of myosin VI-positive dots are moving in and out of the polar region around the spindle poles and around the perinuclear region adjacent to the cleavage furrow (Figure 3C and Supplemental Movie 3). In some cells, myosin VI-positive dots were observed moving on a fixed possible cytoskeletal track between the polar region and the cleavage furrow (Supplemental Movie 4). Unfortunately, we were unable to investigate the nature of this very intriguing but delicate track, because we were unable to visualize this track in cells after fixation. During late cytokinesis when the intercellular bridge is lengthening myosin VI-positive spots now move predominantly away from the midbody into the cell (Supplemental Movie 5). At this time myosin VI may transport endocytic vesicles out of the cytoplasmic bridge and into the cell, because it was recently reported (Schweitzer et al., 2005) that endocytosis resumes in late cytokinesis in the midbody region.
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The Myosin VI Tail Is Not Targeted into the Cleavage Furrow
Previously, we showed that the intracellular localization of myosin VI to vesicular structures in the endocytic and secretory pathways requires the C-terminal cargo-binding domain (Buss et al., 2001; Warner et al., 2003; Sahlender et al., 2005). The targeting of myosin VI to membrane compartments is mediated by several different binding partners or by interaction with phospholipids (Spudich et al., 2007). Whereas the tail domain is sufficient for targeting to clathrin-coated structures at the plasma membrane, the recruitment of myosin VI into membrane ruffles at the leading edge of moving cells also requires its motor domain (Buss et al., 1998).
To establish whether the tail domain of myosin VI is sufficient for targeting into the cleavage furrow during cytokinesis, we compared the localization of full-length GFP-myosin VI and the GFP-tail in a double-labeling experiment with a polyclonal antibody to nonmuscle myosin II to visualize the contractile ring, because we observed that both myosin II and actin are concentrated in the intercellular bridge of MDCK cells (Figures 1 and 4 and Supplemental Figure 3). Whereas the full-length myosin VI shows very good colocalization with myosin II in the contractile ring in the ingressing cleavage furrow (Figure 4, a–c) and afterward in the midbody region (Figure 4, d–f), the GFP-tail showed no cytokinesis-specific labeling pattern (Figure 4, g–i and j–l). The tail domain was targeted to punctate, vesicular structures in dividing cells (Figure 4, g and j), which were often concentrated in the polar regions surrounding the spindle poles (Figure 7g), but no colocalization with myosin II in the cleavage furrow or the midbody region was observed (Figure 4, g and h, j and k).
Inhibition of Myosin VI Activity Delays Mitotic Progression and Leads to Cytokinesis Defects
To probe the function of myosin VI during cell division, we used two different approaches. First we inhibited myosin VI activity by overexpressing a dominant-negative tail mutant of myosin VI in MDCK cells. We used MDCK cell lines that have been generated and characterized recently (Au et al., 2007) and in which protein expression can be induced by addition of ZnCl2. A dominant-negative approach has been used successfully to inhibit myosin VI-dependent functions in the endocytic and exocytic pathway (Buss et al., 2001; Swiatecka-Urban et al., 2004; Au, 2007). To quantify defects in cytokinesis caused by overexpression of the dominant-negative tail mutant, we induced expression of the myosin VI wild-type tail, myosin VI tail mutant (with a mutation RRL->AAA that affects GIPC binding) or full-length myosin VI for 24–36 h, and we compared the number of multinucleated cells with wild-type MDCK cells (control) (Figure 5A). Only overexpression of the dominant-negative wild-type tail gave a significant and consistent eightfold increase in the number of multinucleated cells (<1–8%), thereby demonstrating an increase in cytokinesis failure. The mutant myosin VI tail, however, with the amino acid change (RRL to AAA; aa 1107–1109) that abolishes optineurin and GIPC binding is no longer able to block GIPC-mediated myosin VI function; therefore, it does not cause any increase in multinucleation (Figure 5A). As a second approach to manipulate myosin VI function, we used siRNA oligonucleotides to silence myosin VI expression. For these experiments, we changed from MDCK to HeLa cells, which give much higher siRNA transfection rates, and siRNA SMARTpools are easily obtainable for all human genes. These cells were transfected with a SMARTpool containing four different siRNAs specific for myosin VI. After transfection myosin VI, expression levels were down to <10% as demonstrated by Western blotting (Figure 5B). When compared with mock-treated control cells siRNA treatment did not affect cell viability significantly and the mitotic index of control and KD cells was 2% (data not shown). Depletion of myosin VI from HeLa cells by using the SMARTpool siRNA led to a significant increase in the number of bi- and multinucleated cells (13%) compared with mock-transfected cells (3%), indicating a four- to fivefold increase in cells with defects in cytokinesis (Figure 5C). To exclude off-target effects, we performed rescue experiments in a stable HeLa cell line expressing siRNA-resistant myosin VI, and we observed 4.5% multinucleated cells when these cells were mock transfected and 10% multinucleated cells when transfected with myosin VI siRNA. This indicates only a twofold increase in multinucleation compared with wild-type HeLa cells, where the loss of myosin VI leads to a more than fourfold increase in multinucleation. Therefore, the expression of siRNA-resistant myosin VI leads to a 50% rescue of the cytokinesis defect (Figure 5D).
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Myosin VI Mediates the Insertion of TfR-containing Endosomes into the Midbody Region
The insertion of membranes from the endocytic and secretory pathway into the cleavage furrow is crucial for successful completion of cytokinesis. Because myosin VI has been localized in clathrin-coated and -uncoated endocytic structures (Buss et al., 2001) (Aschenbrenner et al., 2003), we next examined whether myosin VI is involved in delivery of endocytic material into the cleavage furrow. In MDCK cells expressing GFP-myosin VI or GFP-myosin VI tail, we labeled the endocytic compartment with an antibody to the transferrin receptor. This receptor is taken up into mammalian cells via clathrin-mediated endocytosis and after passing through a recycling endosome, it is trafficked back to the cell surface or into the midbody region. GFP-myosin VI and GFP-myosin VI tail both showed very good colocalization with the transferrin receptor positive compartment, possibly the recycling endosome, in the region around the spindle pole (Figure 7, a–c, and g–i, arrowheads). However, in the midbody region, we only observed colocalization between full-length myosin VI and the TfR (Figure 7 a–c, arrow), because in cells expressing GFP-myosin VI tail there was no TfR and very little myosin VI tail was detected in the midbody region (Figure 7, g–i). Our confocal images also demonstrated limited but significant colocalization of GFP-myosin VI with TfR positive endosomes in close vicinity to the midbody region (Figure 7, d–f, arrowheads), which might represent the myosin VI-positive vesicle population moving into and out of the cytoplasmic bridge that we observed in our live cell studies. These results indicate that the myosin VI tail domain is able to inhibit as a dominant-negative mutant the transport of TfR-positive endocytic vesicles into the midbody region.
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GIPC Colocalizes with Myosin VI in the Midbody Region and Loss of GIPC Results in Increased Number of Multinucleated Cells
Targeting of myosin VI to distinct intracellular compartments requires binding to several different binding partners. The study by Skop et al. (2004) indicated that not only myosin VI but also GIPC, a myosin VI binding partner, is present in isolated midbodies. GIPC, a small PDZ domain-containing protein, colocalizes with myosin VI on endocytic vesicles (Aschenbrenner et al., 2003), and it has been shown to bind to a number of transmembrane receptors at the cell surface and the Golgi complex in interphase cells (Katoh, 2002). Using polyclonal antibodies to GIPC, we observed a striking localization of GIPC on either side of the midbody region colocalizing with GFP-tagged myosin VI in MDCK cells (Figure 8, d–f). There was a perfect overlap between myosin VI and GIPC in the midbody region and on vesicular structures on either side of the cleavage furrow (Figure 8, g–i). In addition, both proteins were recruited to the walls of the cleavage furrow during late anaphase/telophase (Figure 8, a–c). These results strongly suggest that a complex between myosin VI and GIPC may be essential for myosin VI to function during cytokinesis. This finding is supported by the observation that the myosin VI tail, where the GIPC binding site is mutated (RRL to AAA) (Spudich et al., 2007) has no dominant-negative inhibitory effect on the function of myosin VI during cytokinesis (Figure 5A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Overall the role of actin filaments in chromosome capture and congression may not be restricted to large oocytes; it also may operate in other smaller somatic cells undergoing mitosis. In these cells, an increasing number of actin-regulating proteins, such as the Rho GTPase cdc42 and its effector mDia, have been shown to be important for cell cycle progression (Yasuda et al., 2004). These proteins induce actin filament nucleation and polymerization and they regulate microtubule attachment to kinetochores and chromosome alignment at the metaphase plate. Therefore, it may not come as a surprise that myosin VI, a unique actin-based motor protein, is recruited to centrosomes/spindle poles during prophase.
However, the loss of myosin VI in knockdown cells does not lead to a complete mitotic arrest. In addition in fibroblasts isolated from the myosin VI knockout mouse (Snell's waltzer mouse), no major defects in chromosome alignment or spindle morphology were observed. These results suggest that redundant pathways exist for spindle assembly and chromosome congression in mammalian cells. The prometaphase delay in myosin VI knockdown cells is very similar to the delay described recently for Clip 170 KD cells (Dujardin et al., 1998; Tanenbaum et al., 2006). Clip 170 was identified as a myosin VI binding partner in Drosophila (Lantz and Miller, 1998); however, in mammalian cells we have not been able to confirm by yeast two-hybrid or mammalian two-hybrid assays or by coimmunoprecipitation any interaction between myosin VI and Clip 170. So far, we were only able to demonstrate direct binding of Clip 170 to myosin VI in a glutathione transferase pull-down assay using recombinant GST tagged myosin VI tail and in vitro expressed Clip 170 (data not shown).
The midbody has a complex multicomponent structure that forms between dividing cells during cytokinesis. A recent proteomics study identified myosin VI and GIPC as mammalian midbody proteins and highlighted a role for these two proteins in germline cytokinesis in C. elegans (Skop et al., 2004). We have confirmed the localization of GIPC in the midbody region in mammalian cells; furthermore, we have shown that in siRNA knockdown experiments, the loss of GIPC leads to a dramatic failure in cytokinesis, resulting in binucleated and multinucleated cells. Detailed immunolocalization studies show that GIPC is concentrated on either side of the midbody, and in double-labeling experiments, GIPC shows a dramatic overlap with myosin VI in the cleavage furrow on either side of the midbody.
Membrane trafficking is an essential part of cytokinesis for delivering and regulating the addition of new membrane along the ingressing cleavage furrow (Strickland and Burgess, 2004; Albertson et al., 2005). The importance of membrane fusion events during cytokinesis is highlighted by the requirement of soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (Low et al., 2003). The new membrane delivered into the cleavage furrow may be derived from the secretory, endocytic, or recycling pathways. In some cell systems, brefeldin A inhibits cytokinesis, indicating that Golgi-derived membrane trafficking is involved (Skop et al., 2001). Furthermore, proteomic analysis of mammalian midbodies identified a number of Golgi-derived proteins (Skop et al., 2004). Because myosin VI plays a role in exocytic membrane trafficking pathways, it could be involved together with its binding partner optineurin in transporting Golgi-derived vesicles into the midbody region (Warner et al., 2003; Sahlender et al., 2005). However, so far we have not observed any colocalization of myosin VI with any Golgi fragments in dividing cells or any targeting of optineurin into the cleavage furrow during cytokinesis. Furthermore, depletion of optineurin by siRNA does not result in a cytokinesis defect and multinucleation. These results indicate that myosin VI is apparently not involved in the transport of secretory vesicles into the midbody area.
Endocytosis and membrane recycling are, however, crucial for cytokinesis, because proteins essential for these processes, such as clathrin, caveolin, dynamin, Arf6, and Rab11, are required (Kogo and Fujimoto, 2000; Gerald et al., 2001; Schweitzer and D'Souza-Schorey, 2002). Recently, it has been shown that in early telophase, endocytosis resumes at the polar regions of the dividing cell (Schweitzer et al., 2005). In this study, it was demonstrated that endocytosed transferrin accumulates first in vesicles around the spindle poles, and then these vesicles are transported to the cleavage furrow in late cytokinesis. Given the localization of myosin VI in vesicles surrounding the spindle poles and in vesicles moving into the midbody region, myosin VI is most likely to be a driving force for the transport of vesicles from the pericentriolar vesicular compartment into the cleavage furrow, because inhibition of myosin VI function dramatically reduces the amount of TfR-containing vesicles transported into the midbody region. The tail domain of myosin VI targets to vesicles in the pericentriolar region but not to the cleavage furrow, indicating that the dominant-negative tail inhibits the formation and/or transport of vesicles away from the pericentriolar recycling/sorting endosome. Although the exact nature of this pericentriolar compartment needs to be established, our previous work has shown that ablating myosin VI function in polarized MDCK cells perturbs the correct sorting of cargo to the basolateral domain (Au et al., 2007). This sorting step is thought to involve the transferrin-positive recycling endosome, which may be a very similar compartment to that in the pericentriolar region in cells undergoing cytokinesis.
In the final stages of cytokinesis, our live cell movies indicate that myosin VI-containing vesicles not only move into the cleavage furrow but also predominately move away from the midbody back into the cell. Because work by Schweitzer et al. (2005) has shown that endocytosis resumes from the midbody region at the end of cytokinesis, it is likely that at this stage myosin VI may be transporting endocytic membranes back into the cell.
During cytokinesis, actin filaments assemble not only as a circumferential belt as part of the contractile ring but also align themselves along the spindle axis in a complex three-dimensional network (Fishkind and Wang, 1993). Although the polarity of these actin filaments is not known, there is an obvious need for a reverse-directed motor such as myosin VI; however, at present, its precise function has not been identified.
The movement of myosin VI to the centrosome in early mitosis and into the cleavage furrow and the midbody region seems to involve interaction with specific binding partners such as GIPC. A minor mutation (RRL->AAA) prevents GIPC binding to the myosin VI tail (Spudich et al., 2007), and it abolishes the ability of the tail to cause cytokinesis defects. Targeting of myosin VI to the ingressing cleavage furrow might also involve its high-affinity binding site for PIP2 in the C-terminal tail domain (Spudich et al., 2007), which we have recently identified. Several studies have recently shown that PIP2 accumulates at the cleavage furrow and that its presence is necessary for successful furrow ingression during cytokinesis (Emoto et al., 2005; Field et al., 2005; Janetopoulos et al., 2005; Janetopoulos and Devreotes, 2006).
The dual localization of myosin VI in dividing cells at and around the centrosome in early mitosis and later in the midbody region during cytokinesis is very intriguing, and future experiments will be required to determine whether myosin VI displays separate independent functions during prometaphase at the centrosome and during cytokinesis or whether it is part of the signaling networks that coordinate or regulate mitosis, cytokinesis, and abscission.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Folma Buss (fb1{at}mole.bio.cam.ac.uk).
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