Report |
Address correspondence to Ryoko Kuriyama, Dept. of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, 321 Church St. SE, University of Minnesota, Minneapolis, MN 55455. Tel.: (612) 624-0471. Fax: (612) 626-6140. E-mail: ryoko{at}lenti.med.umn.edu
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Abstract |
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Key Words: actin; alternative splicing; cytokinesis; kinesin-like protein; midbody
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Introduction |
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MKLP1/CHO1-related proteins show dynamic changes in their subcellular distribution during the cell cycle. In mammalian cells, CHO1 is present in interphase centrosomes and nuclei and becomes associated with the mitotic spindle. As chromosomes move toward poles, the protein shifts to the midzone and eventually concentrates into a bright spot in the middle of the intercellular bridge (Sellitto and Kuriyama, 1988). Since it is a plus-enddirected motor present in the interzonal region of the spindle, MKLP1/CHO1 was originally thought to function in chromosome separation and spindle elongation during karyokinesis (Nislow et al., 1992). In fact, microinjection of CHO1 antibodies caused mitotic arrest in mammalian cells (Nislow et al., 1990) and sea urchin embryos (Wright et al., 1993). However, genetic analysis has suggested involvement of zen-4 and pavarotti in cytokinesis rather than karyokinesis (Adams et al., 1998; Powers et al., 1998; Raich et al., 1998). Evidence has also been presented that overexpression of the mutant CHO1 and RNA-mediated interference specifically blocked completion of cytokinesis in mammalian cells (Matuliene and Kuriyama, 2002). The ability of the motor proteins to organize central spindles and the midbody appears to be essential for their function in cytokinesis, which could be achieved through their interaction with Aurora kinase AIR-2 (Schumacher et al., 1998; Severson et al., 2001) and RhoGAP Cyk-4 (Jantsch-Plunger et al., 2000).
To clarify the nature of molecular diversity among species and define the role of MKLP1/CHO1 during cell division, we examined the chicken genomic sequence. Here we report that heterogeneity of the COOH-terminal tail is derived from alternative splicing and that the sequence encoded by exon 18 is expressed in only the CHO1 isoform. Exon 18 includes a polypeptide capable of interaction with F-actin in vivo and in vitro, and microinjection of exon 18specific antibodies blocked the terminal phase of cytokinesis. A possibility of CHO1 involvement in membrane events, rather than the actin-containing contractile ring in the cell cortex, is discussed.
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Results and discussion |
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CHO1-specific exon 18 encodes an actin-interacting domain
To characterize the CHO1-specific sequence, we expressed 91 amino acids encoded by exon 18 in CHO cells. The green fluorescent protein (GFP)-tagged exogenous polypeptide is located inside the cytoplasm in association with cytoskeletal fibers (Fig. 4 A). Double staining with Alexa-phalloidin (MFs) provided evidence that the fibers to which the exon 18 sequence attaches are actin filaments. When cells were treated with dihydrocytochalasin B, GFP was no longer detected along cytoskeletal fibers (Fig. 4 B, +DHCB). Instead, actin-containing dots formed by depolymerization of F-actin become visible by GFP fluorescence. In contrast, the exon 18 polypeptide does not reveal any affinity to the microtubule network (Fig. 4 C). This is in good agreement with our previous observation that CHO1 interacts with microtubules through microtubule-binding sites located at the NH2-terminal half of the protein (Matuliene and Kuriyama, 2002).
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CHO1 is seen inside the nucleus, which is due to the presence of a nuclear localization signal at the COOH-terminal end of the tail (unpublished data). When the full-length CHO1 lacking nuclear localization signal was expressed in interphase cells, the tagged molecule was located in the cytoplasm in association with both microtubules and F-actin (unpublished data). However, in mitotic cells the protein was seen predominantly in the spindle, and virtually no fluorescent signal was detected in the cortex where the actin-containing contractile ring is assembled. Since the exon 18 sequence was targeted to the cortex, especially in the vicinity of the furrow during cytokinesis in mitotic cells (unpublished data), the interaction of CHO1 with actin-containing structures may be controlled in a cell cycledependent manner.
Cells treated with E18 antibodies failed to complete the terminal phase of cytokinesis
By mutating the mechanochemical motor domain of CHO1, we have shown previously that the protein is required for completion of cytoplasmic division in mammalian cells (Matuliene and Kuriyama, 2002). To examine whether inhibition of the actin-interacting domain results in the similar phenotypes, we microinjected the affinity purified E18 antibody into PtK1 cells. In Fig. 5, the metaphase cell received E18 at time zero (Fig. 5 A) underwent normal chromosomes separation (Fig. 5 C) and cytoplasmic division (Fig. 5 D). Although two daughter cells appeared to be separated completely, the cell boundary became unclear (Fig. 5, E and F), and the two cells eventually merged together by 3 h after antibody injection (Fig. 5 G). mAb staining of the fixed cell clearly indicated the formation of a midbody between two separated nuclei (Fig. 5, arrows). Nonetheless, the furrow ultimately resumed to produce a binucleate cell (Fig. 5, DAPI), suggesting that E18 specifically inhibits the terminal phase of cytokinesis. The effect of E18 was specific because no major inhibition of cell division was observed in cells treated with injection buffer alone, commercially available nonspecific rabbit IgG, and protein Apurified E18 preimmune antibodies (Table I). These results contrast with those of Nislow et al. (1990) and Wright et al. (1993) who observed mitotic arrest in mAb-injected cells. The difference may be attributed to the specificity of the antibodies injected into cells (mouse monoclonal IgM recognizing the central stalk versus rabbit E18 IgG specific to the tail). The role of MKLP1/CHO1 in early stages of mitosis is left unsolved.
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Alternatively, the motor protein could be involved in membrane events necessary for completion of cytokinesis. It is widely believed that targeted secretion, insertion, and fusion of membrane vesicles play a central role in progression and completion of cleavage furrows (for review see Straight and Field, 2000). Thus, cells lacking syntaxins and their associated proteins (Lukowitz et al., 1996; Heese et al., 2001), a phospholipid kinase (Brill et al., 2000), dynamin (Gu and Verma, 1996), or Golgi-associated proteins (Sisson et al., 2000), are defective in cytokinesis in a variety of cells. Rabkinesin-6/Rab6-KIFL, a Rab6-binding kinesin-like protein, is required for not only membrane traffic but also cytokinesis (Hill et al., 2000). Of particular interest is that this motor protein shares the highest degree of sequence identity to CHO1 (Echard et al., 1998). Skop et al. (2001) have reported recently that brefeldin, a potent inhibitor of vesicle secretion by targeting a small GTPase-binding protein Arf, specifically blocks the terminal phase of cytokinesis by regressing ingressed furrows in C. elegans. The phenotype detected in brefeldin-treated embryos is remarkably similar to what we saw in E18-injected mammalian cells. Importantly, CHO1/MKLP1 is capable of binding Arfs through the sequence encoded by exons 1922, just downstream the actin-interacting domain of exon 18 (Boman et al., 1999). Since dividing cells contain dynamic membrane phospholipids tightly coupled with the actin cytoskeleton (Emoto and Umeda, 2000) and Arfs are believed to be involved in both membrane traffic and actin dynamics, it is likely that the actin- and Arf-interacting domains act in concert to achieve the CHO1 function during the late stage of cytokinesis. Further analysis of CHO1 would be of great benefit for our understanding of the mechanism and regulation of cell division.
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Materials and methods |
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Cell culture, protein expression, and immunostaining
CHO and HeLa cells were cultured in 10% FCS containing Ham's F-10 and DME medium, respectively. The exon 18 coding sequence (amino acid positions 696786) was isolated by digestion of the full-length CHO-CHO1 with SspI and Eam1104 I and ligated into the eukaryotic expression vector, pEGFP-C1 (CLONTECH Laboratories, Inc.). CHO cells on a coverslip in a 35-mm dish were transfected by addition of 0.62 µg/ml purified plasmid DNA and cultured overnight (Matuliene and Kuriyama, 2002). To enrich mitotic cell populations, cells were partially synchronized by treatment with thymidine followed by accumulation at M phase by addition of nocodazole at a final concentration of 0.05 µg/ml (Sellitto and Kuriyama, 1988). After washing out the drug, cells were allowed to recover for 2050 min before fixation with cold methanol.
For immunofluorescence staining, cells were rehydrated with 0.05% Tween-20 containing PBS and incubated with primary and secondary antibodies. Primary antibodies include monoclonal anti-tubulin antibodies (Sigma-Aldrich), monoclonal CHO1 (mAb [Sellitto and Kuriyama, 1988]), and rabbit polyclonal antibodies (E18) raised against 23 amino acids in exon 18 at amino acid positions 755777 of chicken CHO1 (Fig. 2, double underlined). Immunoblotting analysis was done as described before (Kuriyama et al., 1994).
In vitro cosedimentation with F-actin
Actin was prepared from rabbit skeletal muscles (a gift from Drs. Albina Orlova and Ewa Prochniewicz, University of Minnesota, Minneapolis, MN) (Orlova et al., 1995), and platelet actin was purchased from Cytoskeleton, Inc. G-actin was polymerized and sedimented in a medium containing 5 mM Tris-HCl, pH 7.8, 0.2 mM ATP, 0.1 mM CaCl2, 150 mM KCl, and 1 mM MgCl2. For protein expression in insect cells, cDNA encoding GFPexon 18 was subloned into pVL1392 (PharMingen) and used for infection of Sf9 cells as before (Kuriyama et al., 1994). 35S-labeled GFPexon 18 was synthesized using a commercially available kit (TNT Coupled Reticulocyte Lysate System; Promega). CHO and Sf9 cells expressing exogenous proteins were washed once with PBS and lysed for 3060 min at 0°C in a medium containing 10 mM Tris-HCl, pH 7.8, 0.5% Nonidet NP-40, and protease inhibitors. Cell extracts and reticulocyte lysates recovered after centrifugation at 200,000 g for 30 min were mixed with F-actin and further incubated for 30 min on ice. After layering on a 20% sucrose cushion, F-actin plus associated proteins were sedimented at 100,000 g for 30 min at 4°C.
Antibody injection
Affinity purified E18 was prepared in injection buffer (140 mM KCl, 100 mM glutamic acid, 40 mM citric acid, 1 mM MgCl2, 1 mM EGTA, pH 7.4) and injected into PtK1 cells cultured on a photoetched coverslip (Bellco). Microinjection was performed using a Narishige microinjector attached to a Nikon Diaphot inverted microscope, and time-lapse images were recorded with a Nikon Eclipse TE200 microscope using ImagePro software packages.
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Footnotes |
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Acknowledgments |
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Submitted: 24 September 2001
Revised: 18 January 2002
Accepted: 18 January 2002
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References |
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