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© The Rockefeller University Press,
0021-9525/2000//499 $5.00
The Journal of Cell Biology, Volume 150, Number 3,
, 2000 499-512
Original Article |
A Kinesin-Related Protein, Krp180, Positions Prometaphase Spindle Poles during Early Sea Urchin Embryonic Cell Division
jmscholey{at}ucdavis.edu
We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP180, in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP180 concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP180 is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.
Key Words: Xklp2 • kinesin • sea urchin • mitosis • 
-tubulin
© 2000 The Rockefeller University Press
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Introduction |
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We have used cleavage stage echinoderm embryos to characterize the functions of mitotic motors, including motors that are involved in spindle pole positioning. The living echinoderm embryo is a classic developmental system for studies of mitosis and cell division as it offers excellent cytology and is amenable to micromanipulation (Wright et al. 1993; Morris and Scholey 1997; Scholey 1998). Members of two families of MT-based motors, the kinesins and dyneins, are known to function in positioning both chromosomes and spindle poles during mitosis in a variety of systems (Goldstein 1993; Vaisberg et al. 1993; Holzbaur and Vallee 1994; Vernos and Karsenti 1995; Echeverri et al. 1996; Karsenti et al. 1996; Starr et al. 1998; Sharp et al. 2000). In sea urchin embryos, experiments including microinjection of pan-kinesin antibodies revealed critical mitotic functions for kinesin-related motors in general (Wright et al. 1993). Furthermore, the microinjection of class-specific antibodies revealed that two kinesin-related protein (KRP) motors, KRP110 and KRP170, are required for spindle assembly and function, presumably by acting to position spindle poles (Wright et al. 1993; Chui, K.K., G.C. Rogers, A.M. Kashina, K.P. Wedaman, D.J. Sharp, D.T. Nguyen, and J.M. Scholey, manuscript submitted for publication). Finally, a calmodulin-binding COOH-terminal kinesin, Kinesin-C, is present in the early embryo where it is also suspected to participate in spindle pole positioning, but, so far, its function has not been experimentally tested (Rogers et al. 1999).
The functional inhibition of a number of different mitotic motors that are believed to position spindle poles in several systems results in a strikingly similar terminal phenotype: monoastral spindle formation, characterized by duplicated, but closely spaced, spindle poles surrounded by a MT aster attached distally to chromosomes (Vernos et al. 1995; Kashina et al. 1997; Molina et al. 1997; Sharp et al. 1999). Monoastral spindles can arise by one of two pathways: (a) a failure of duplicated spindle poles to separate during spindle assembly, or (b) a plateward collapse of separated poles due to a failure in maintaining spindle shape. Such a phenotype is observed by perturbing the function of the Xenopus laevis kinesin Xklp2 in vitro (Boleti et al. 1996). Xklp2 is a plus end–directed kinesin that localizes to centrosomes throughout the cell cycle, being localized to MT minus ends through its interaction with an accessory protein, TPX2 (targeting protein for Xklp2; Wittmann et al. 1998). Functional inhibition of Xklp2 before or after spindle assembly in Xenopus egg extracts results in the formation of monoastral spindles (Boleti et al. 1996), leading to the proposal that Xklp2, tethered near the centrosome, moves toward the plus ends of MTs attached to the opposite centrosome, thereby driving the separation of spindle poles during spindle assembly and maintenance.
In this report, we have identified and characterized a homologue of Xklp2, called KRP180, from sea urchin early embryos. Using quantitative, real-time imaging of embryos microinjected with inhibitors of KRP180, we show that this motor plays a critical role in positioning spindle poles during mitosis, specifically at prometaphase. Immunolocalization of KRP180 and sea urchin -tubulin reveals that this motor is not a component of the centrosome but instead concentrates in the region of the central spindle where it may position spindle poles by generating forces on microtubules that are attached to the poles. This study is the first functional analysis of the mitotic function of a member of the Xklp2 subfamily in a living system.
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Materials and Methods |
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Cloning Sea Urchin KRP180 and -Tubulin Isoforms
A PCR screen of a S. purpuratus unfertilized egg 
ZAP library (Stratagene) with hyperconserved kinesin motor domain sequences yielded a partial cDNA clone encoding the KRP180 motor domain. This clone was used for additional library and PCR screens (Rogers et al. 1999) that identified a total of five different KRP180 cDNAs that collectively span the entire 4,392-bp ORF.
A partial cDNA encoding sea urchin -tubulin was identified in a nested PCR screen of the egg cDNA library using four degenerate primers to hyperconserved sequences found in higher eukaryotic -tubulins (Rogers 2000). This original clone (c9), encoding SpGamma1, was then used in a library screen and two additional -tubulin cDNAs were identified [c1 (1.38 kb) and c3 (0.94 kb)] whose nucleotide sequence were identical to each other but different from c9. These two new clones encode a different -tubulin isoform (SpGamma2); c1 encodes the entire SpGamma2 ORF. Sequence analysis and structural predictions were performed using the GCG sequence analysis software (Devereux et al. 1984) unless otherwise stated.
Recombinant Proteins and Antibody Production
Two expression plasmids for a GST fusion with the KRP180 NH2-terminal stalk region (named GST-Coil 1) and COOH terminus (named GST-Coil 2) were made by directionally subcloning KRP180 nucleotides 1,191–1,965 and 3,495–4,389 into pGEX-GST (Amersham Pharmacia Biotech). E.coli–expressed GST-Coil 2 was purified under nondenaturing conditions on glutathione-agarose (Amersham Pharmacia Biotech) and then injected into four female BALB/c mice. After an adequate immune response was reached, polyclonal ascites formation was induced by injection of T-180 sarcoma cells (ATCC) intraperitoneally. Four different batches of ascites fluid from the four mice are identified as polyclonal antibodies 180.1, 180.2, 180.3, and 180.4.
A full-length SpGamma2 cDNA was PCR amplified, subcloned directionally into a pGEX-GST expression plasmid, and expressed/purified under nondenaturing conditions as described for GST-Coil 2. Anti-human -tubulin peptide antibody (catalog no. T3559; Sigma-Aldrich) was affinity-purified against recombinant GST-SpGamma2 protein.
Polyclonal antibodies from the anti-KRP180 (180.1-180.4) and T3559 sera/crude IgG were affinity-purified against Affigel (Bio-Rad Laboratories) columns precoupled to their respective antigens (GST-Coil 2 and GST-SpGamma2 protein). All affinity-purified antibodies were acid eluted, neutralized in Tris buffer, dialyzed into either microinjection buffer (MIB: 150 mM K-aspartate and 10 mM K-phosphate, pH7.2) or TBS, and concentrated.
Hydrodynamic Studies of KRP180
Native KRP180 was partially purified from S. purpuratus egg cytosolic extract by a cycle of binding to, and subsequent low salt/ATP elution from, endogenous taxol-stabilized MTs (see Chui, K.K., G.C. Rogers, A.M. Kashina, K.P. Wedaman, D.J. Sharp, D.T. Nguyen, and J.M. Scholey, manuscript submitted for publication). To determine the Stokes radius and sedimentation coefficient of KRP180, MT elution fractions were evenly divided and placed over a gel filtration column or on linear 5–20% sucrose density gradients and all subsequent fractions screened for KRP180 by Western blot. The native molecular mass of KRP180 was calculated by the method of Siegel and Monty 1966 with the partial specific volume adjusted to 0.716 (Zamyatnin 1972).
Immunofluorescence Microscopy
Fixation and pre-extraction of sea urchin embryos were performed as previously described (Henson et al. 1995). In brief, dejellied eggs were washed in filtered natural sea water (FNSW) containing 10 mM PABA, fertilized, and monitored with a dissection microscope until first cleavage. Fertilization envelope stripped embryos were fixed with 100% methanol (–20°C), rehydrated stepwise into methanol solutions of TBS + 0.05% Tween (TBST), and blocked with 5% normal goat serum (NGS).
Pre-extraction of mitotic embryos was performed using either 1% NP-40 or 1% Triton X-100 detergent for either 10 or 15 min. Affinity-purified anti-KRP180 antibody was used at 1 µg/ml in TBST + 5% NGS. Two different anti–
/β-tubulin antibodies were used: an anti-bovine brain tubulin (Gelfand lab) and an FITC-conjugated anti–-tubulin monoclonal DM1a (Sigma-Aldrich). Double-labeling with the DM1a and anti-KRP180 antibodies was performed as described in Rogers 2000. Secondary antibodies were purchased from Jackson ImmunoResearch Lab. Inc. DAPI (1 µg/ml) was added to the embryos and washed extensively before mounting on poly-L-lysine–coated glass coverslips in a 90% glycerol solution containing 20 mg/ml phenylenediamine in PBS. Indirect immunofluorescent micrographs were obtained as either single optical sections or projections using a Leica TCS SP confocal with each image resulting from 16–32 averaged scans and analyzed using Adobe Photoshop v5.0.
Antibody and Recombinant Protein Microinjection
Control mouse IgG or the four pooled anti-KRP180 polyclonal antibodies (180.1–180.4), were dialyzed into MIB and concentrated to 12.4 mg/ml (final intracellular [Ab] at 0.62 mg/ml). Glutathione–Sepharose-purified recombinant GST, GST-Coil 1, and GST-Coil 2 protein was purified in MIB buffer, concentrated, and microinjected at a range of concentrations (final intracellular [protein] from 0.96–9.1 µM). We observed an identical consistent phenotype after introducing GST-Coil 2 protein into early embryos regardless of the protein concentration used, suggesting that the lowest concentration of 0.96 µM was sufficient to inhibit KRP180 function. Embryo microinjections were performed by a procedure modified from Kiehart 1982 and Wright et al. 1993. Lateral injection chambers (Kiehart 1982) allowed high-resolution observation of injections and development by restraining the embryos during cleavage until the ciliated blastula stage. Fertilized eggs were injected with 5% cell volume between 20 min after fertilization until cytokinesis of first cleavage. Embryos were observed by differential interference contrast microscopy on an inverted microscope (model IM-35; Carl Zeiss, Inc.) using a Plan 40x objective and imaged as described (Wright et al. 1993; Morris and Scholey 1997). An Argus 10 Image Processor (Hamamatsu Photonics) was used for contrast enhancement.
Quantitation of spindle pole positioning and morphology was performed by capturing video printouts of injected embryos, using a Sony UP-880 video graphic printer, every 0.5–5 min at the time of nuclear envelope breakdown (NEBD) or after injection if a spindle was already present. Video prints were digitized using a flatbed scanner and processed using Adobe Photoshop v5.0 and NIH Image v1.62. Spindle pole-to-pole distances were measured as the distance between the center-points of each spindle pole. Changes in these movements over time were plotted using Cricket Graph v1.3.2.
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The deduced primary sequence of KRP180 predicts a polypeptide 1,463 amino acids in length and a molecular mass of 166,590 Daltons (Fig. 1 A), with an NH2-terminal motor domain (amino acid residues 1–347) linked to an extensive segment of predicted -helical coiled-coil, 106 nm long (residues 362-1463; Fig. 1 C). The presence of two predicted PEST sequences (at residues 393–426 and 709–742 with PEST scores +13.22 and 7.56, respectively) suggests that this protein is a target for proteolysis and rapid turnover (Rogers et al. 1986). In addition, two p34cdc2 kinase phosphorylation sites [(T/S)PX(K/R); Nigg 1993; Fig. 1 A], and a predicted tyrosine kinase phosphorylation site (residues 1,135–1141) were found in the stalk region.
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Native KRP180 from Sea Urchin Eggs Is a Homodimer
To determine the hydrodynamic properties and subunit composition of native KRP180, we raised four mouse polyclonal antibodies against a region of the motor's COOH terminus fused to GST (Fig. 2, lane GST-Coil 2). All four affinity-purified antibodies (180.1–180.4) specifically recognized a 180-kD polypeptide in unfertilized sea urchin egg extract (Fig. 2). Native KRP180 was partially purified from egg extract by a single cycle of AMP-PNP–induced binding to endogenous MTs and subsequent release with ATP in either a high or low salt buffer. This eluate was further fractionated by gel filtration and sucrose density gradient centrifugation to measure the hydrodynamic properties of KRP180 (Table ). KRP180 behaves as a homogeneous protein throughout the biochemical fractionation steps and has a Stokes radius (RS) of 10.07 nm and a sedimentation coefficient of 8.33 S. (Although, in high salt KRP180 displays a sedimentation coefficient of 5.8 S, suggesting that it exists in a folded conformation in low salt that then assumes an elongated conformation in high salt.) Based on these hydrodynamic properties, native KRP180 is calculated to have a molecular mass of 334 kD and an axial ratio of 20–40, indicative of a long rod-shaped molecule. Since the deduced mol wt of the cDNA-encoded polypeptide is 167 kD, we predict the native KRP180 quaternary structure to be a homodimer lacking any accessory proteins.
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-tubulin, a well characterized component of the centrosome.
A PCR screen identified two different isoforms of sea urchin -tubulin: a partial cDNA encoding SpGamma1 and a full-length cDNA encoding SpGamma2 (Fig. 4 A). Both -tubulin isoforms share significant overall identity (72–89%) with Drosophila melanogaster, Xenopus, and human -tubulins (Fig. 4A and Fig. B). We obtained a commercial anti–-tubulin antibody, T3559, because its peptide antigen (residues 38–53 of human -tubulin) differed from a sequence found in SpGamma2 by only two conservative substitutions (both Glu to Asp; Fig. 4 A). After affinity-purification against recombinant GST-SpGamma2 (Fig. 4 C, lane 1), the T3559 antibody specifically recognized a single polypeptide in egg high speed supernatant (HSS) that we assumed to be either one or more maternally loaded -tubulins (Fig. 4 C, lane 2), as well as recombinant GST-SpGamma2 (Fig. 4 C, lane 3). In addition, T3559 antibody stained centrosomes in pre-extracted mitotic embryos (Fig. 4 D). Pre-extraction of one-cell stage metaphase embryos did not affect the bright filamentous staining of KRP180 that colocalized with MTs in the central region of the spindle between the aligned chromosomes (Fig. 5, A–D). However, pre-extraction significantly diminished the punctate staining of KRP180 on the spindle poles; longer periods of pre-extraction eliminated nearly all pole staining (Fig. 5, E–H). In embryos that were pre-extracted for an amount of time where the motor was still associated with the poles, KRP180 did not colocalize with -tubulin at the centrosome (Fig. 5, I–L). The localization of KRP180 to the central region of the spindle where MTs are predicted to overlap into antiparallel arrangements suggests this motor could crossbridge antiparallel MTs to promote MT-sliding.
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33 µm for an average of 14.6 ± 4.3 min (n = 6) before initiating anaphase. During anaphase B, these embryos displayed two distinct phases of spindle pole separation: an initial slow phase lasting an average of 12.2 ± 1.9 min (n = 6) and moving at an average rate of 1.08 ± 0.45 µm/min (n = 5) and a fast second phase (8.8 ± 1.7 min [n = 6], 3.55 ± 0.38 µm/min [n = 5]) (Fig. 7; Table ).
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Of the anti-KRP180 antibody containing embryos that were injected in interphase and entered mitosis, all clearly displayed two separated spindle poles positioned on opposite sides of the nucleus before NEBD, suggesting that KRP180 is not required for the initial separation of spindle poles at prophase (12 of 12 embryos; categories a and c above). However, all of these embryos underwent a dramatic spindle collapse immediately upon NEBD where separated poles moved plateward at an average rate of 2.0 ± 0.41 µm/min (n = 4; Fig. 6 B; Table ). Measurements of spindle pole separation as a function of time of a representative embryo is shown in Fig. 7. Although the spindle had collapsed, poles were clearly visible in a side-by-side position (Fig. 6 B; 15.32 min) with an average pole-to-pole distance of 20.4 µm ± 0.25 (n = 6). Most collapsed spindles continued into anaphase displaying both slow and fast phases of anaphase B at rates and durations observed in controls (8 of 12 embryos; Fig. 7; Table ). However, 4 of 7 embryos did display subtle defects in the rates and length of time spent in the first slow phase of anaphase B, and slight defects in cytokinesis were observed as well (3 of 8).
In addition to antibody microinjection, we studied this motor's mitotic function by introducing recombinant truncated KRP180 proteins into one-cell stage embryos. Such constructs might act as dominant negative proteins by competing with the endogenous motor complex for binding to cargo or transiently-associated accessory polypeptides. To this end, we designed NH2- and COOH-terminal GST-tagged coiled-coil encoding constructs designated GST-Coil 1 and GST–Coil 2 (Fig. 8 A and 2). The sequence for the GST-Coil 1 and Coil 2 domains was based on the work of Boleti et al. 1996 who found that GST constructs of the exactly homologous Xklp2 domains functioned as a negative control and as a dominant negative, respectively, in in vitro spindle assembly assays.
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As was found for control IgG injected embryos, microinjection of either GST-Coil 1 (n = 9) or control GST protein (n = 11) during interphase or mitosis had no deleterious effect on first cleavage embryos. Kinetic analysis of pole-to-pole positioning revealed that GST-Coil 1– and GST-injected embryos maintained average metaphase spindle lengths of 31.8 µm and 30.9 µm, respectively, before initiating anaphase (Fig. 8 B; Table ). Likewise, during anaphase B, these embryos displayed a characteristic biphasic separation of spindle poles: an initial slow phase followed by a fast second phase (Fig. 8 B; Table ).
Thus, the injection of KRP180 inhibitors, either antibody or a dominant negative construct, results in the collapse of bipolar spindles. Taken together, these data suggest that KRP180 functions to position spindle poles and thus to transiently maintain prometaphase spindle shape in early sea urchin embryos.
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KRP180, a Mitotic Motor Required to Maintain Prometaphase Spindle Shape
Our immunolocalization data suggest that KRP180 is localized to interzonal MTs where it could serve to generate forces that position the attached spindle poles during prometaphase. In contrast, Xklp2 concentrates on centrosomes during all cell cycle stages (Boleti et al. 1996). Thus, although both KRP180 and Xklp2 are found to position spindle poles, the mechanisms by which they are proposed to do so differ.
Based on its localization to a region of the spindle where MTs overlap in an anti-parallel fashion, it is tempting to hypothesize that KRP180 functions to crossbridge anti-parallel MTs and drive MT-sliding. Assuming that KRP180 is a plus end–directed motor as was found for Xklp2 (Boleti et al. 1996), it could drive the separation of spindle poles by exerting force on antiparallel spindle MTs to which the poles are attached, as has been suggested with members of the bimC/bipolar kinesin subfamily (Sharp et al. 2000).
Microinjection of sea urchin embryos with KRP180-specific antibodies and recombinant dominant/negative constructs demonstrates that KRP180 is required to maintain the separation of spindle poles during first cleavage. Immediately after NEBD, every anti-KRP180 and GST-Coil 2 injected embryo we observed underwent a dramatic spindle collapse: poles moved plateward at a linear rate and assumed a juxtaposed position, giving rise to monoastral spindles. Such monoastral spindles are a hallmark of cells that fail to divide. For example, similar structures have been seen after the inhibition of either Xklp2 or Eg5 in Xenopus extracts suggesting that both these motors contribute to the formation of bipolar spindles (Sawin et al. 1992; Boleti et al. 1996). However, since the studies in Xenopus were performed using fixed images, it was unclear exactly when these motors exert their effects. In contrast, the live cell imaging described here strongly suggests that KRP180 serves to generate outward forces on spindle poles specifically after the initial separation of the spindle poles during prometaphase.
Quantitation of spindle pole movements in both anti-KRP180 and dominant negative protein injected embryos revealed that most collapsed spindles were able to initiate anaphase and complete mitosis. Occasionally we observed slight defects in spindle elongation and cytokinesis. We attribute these observations to structural defects in the organization of the spindle midzone/midbody on which these events depend, and that perhaps these structures had not assembled properly due to the close proximity of the poles after the spindle collapse. However, due to its midzone/midbody localization, we cannot rule out the possibility that KRP180 might function to assemble or promote MT-sliding during late mitosis in a semi-redundant fashion with other mitotic motors as well (see below).
Proposed Model of Mitotic Motor Functions
How is the mitotic function of KRP180 integrated into the mitotic function of other motors that operate in the early sea urchin embryo? Assuming that the functional inhibition of KRP180 by specific antibody and dominant/negative protein microinjection reveals the full spectrum of this motor's mitotic function, then these results suggest a specific role for KRP180 in maintaining prometaphase spindle shape. In addition to KRP180, we have also characterized new sea urchin motors that play important roles in positioning spindle poles during embryonic cleavage. These include the spindle-associated motors, KRP110 and KRP170 (members of the plus end–directed MKLP1 and bipolar kinesin subfamilies, respectively), a cortically localized cytoplasmic dynein, and a COOH-terminal kinesin, Kinesin-C, that is related to a unique class of Ca2+/calmodulin–regulated motors and whose function is unknown (Chui, K.K., G.C. Rogers, A.M. Kashina, K.P. Wedaman, D.J. Sharp, D.T. Nguyen, and J.M. Scholey, manuscript submitted for publication; Rogers et al. 1999; Rogers 2000). Functional analysis of KRP110, KRP170, and cortical dynein, by microinjection of antibodies and dominant/negative inhibitors, revealed that perturbation of any one of these three motors results in a prophase cell cycle arrest, implying a role for these motors in the initial assembly of the spindle. Similar studies also demonstrated a metaphase or anaphase cell cycle arrest when KRP110 and KRP170 function was perturbed, respectively. In addition, both KRP170 and cortical dynein are required for specific phases of spindle elongation at anaphase.
We propose that the predominant force driving spindle pole positioning is through the MT-sliding activity of these mitotic motors. Native KRP110 and KRP170 are homotetramers with a predicted bipolar ultrastructure (i.e., two motor domains on opposite ends of a coiled-coil rod), whereas native KRP180 is homodimeric with an extensive 106-nm rod. Interestingly, KRP180 has an axial ratio of 20 like KRP110, whereas KRP170 has a longer axial ratio of 80. Given the differences in axial ratios, these motors could crossbridge adjacent MTs with varying space between them. For example, adjacent MTs with a long spacing would be cross-linked by KRP170 that would then be linked into tight MT bundles by the action of KRP110 and KRP180. Such an activity of anti-parallel MT capture and bundling by MT cross-linking motors, combined with their subsequent force generating properties to promote MT sliding, would result not only in the ability to position spindle poles, but to establish the structural integrity of the spindle. Not surprisingly, functional inhibition of these motors results in cell cycle arrest phenotypes, possibly due to direct interference with spindle mechanics. We also propose that minus end–directed, cortical dynein and Kinesin-C function to position spindle poles using a MT-sliding mechanism. COOH-terminal kinesins function to counterbalance the pole separating forces of the bipolar kinesins, whereas dynein slides MTs relative to an actin-rich cortical network to which it localizes (Saunders and Hoyt 1992; Pidoux et al. 1996; O'Connell et al. 1993; Mountain et al. 1999; Sharp et al. 1999, Sharp et al. 2000; Rogers 2000).
These results suggest a plausible model for the pathway of spindle pole positioning during embryonic cell division through the coordinating functions of five different plus and minus end–directed MT-based motors (Fig. 9). Before NEBD, cortical dynein, KRP110, and KRP170 work together to coordinate the initial separation of spindle poles. Pole separation would result from the combined activities of cortical dynein pulling on astral MTs and the homotetrameric (and predicted bipolar) motors, KRP110 and KRP170, cross-linking and sliding anti-parallel MTs. KRP170, which has a greater axial ratio than KRP110, would cross-link adjacent anti-parallel spindle MTs with a long spacing that would then be linked into tighter MT bundles by the action of KRP110, serving to stabilize the connections between the poles as they are pushed/pulled around the nucleus.
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Spindle elongation could be triggered by the inactivation of Kinesin-C. Plant homologues of Kinesin-C are inactivated when their motor domains bind Ca2+/calmodulin (Song et al. 1997). In sea urchin embryos, [Ca2+]i drops after NEBD and then rises globally at the metaphase-anaphase transition (Groigno and Whitaker 1998). In our model, Kinesin-C motor activity is turned on after NEBD when it would crossbridge MTs and maintain spindle shape by exerting a plateward force on the poles throughout (pro)metaphase. When [Ca2+]i increases at the beginning of anaphase, Kinesin-C is turned-off, releasing the plateward force on the spindle poles and allowing spindle elongation to occur in two extensive phases driven by the MT-MT sliding bipolar kinesin, KRP170, and cortical dynein pulling on astral MTs (Fig. 9 C). Additional studies performed in echinoderm embryos (Scholey 1998) combined with complementary studies performed in organisms amenable to genetic manipulation (Sharp et al. 2000) will be required to test this model and elucidate the precise roles that multiple mitotic motors play in spindle pole positioning.
In conclusion, the data described in this paper identify KRP180 as a member of the Xklp2 subfamily of mitotic motors and provide the first evidence of this motor's activity in vivo. A combination of function-blocking reagents was used to show that KRP180 serves to position spindle poles specifically during prometaphase. The notion that this motor functions during such a specific phase of mitosis underscores the subtle roles that some mitotic motors may perform in spindle function.
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health no. GM55507 to J.M. Scholey.
Submitted: 4 February 2000
Revised: 2 June 2000
Accepted: 27 June 2000
Abbreviations used in this paper: DAPI, 4'-6-diamidino-2-phenylindole; GST, glutathione-S-transferase; KRP, kinesin-related protein; MAP, microtubule-associated protein; MTs, microtubules; NEBD, nuclear envelope breakdown; ORF, open reading frame.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Boleti H. Karsenti E. Vernos I. Xklp2, a novel Xenopus centrosomal kinesin-like protein required for centrosome separation during mitosis, Cell, 84, 1996, 49–59.[Medline]
Chui, K.K., G.C. Rogers, A.M. Kashina, K.P. Wedaman, D.J. Sharp, D.T. Nguyen, and J.M. Scholey. 2000. Roles of two kinesins in sea urchin embryonic cell division. Submitted.
Devereux J. Haeberli P. Smithies O.. A comprehensive set of sequence analysis programs for the VAX, Nucleic Acid Res, 12, 1984, 387–395.
Echeverri C.J. Paschal B.M. Vaughan K.T. Vallee R.B.. Molecular characterization of the 50-kd subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis, J. Cell Biol., 132, 1996, 617–633.
Goldstein L.S.B.. With apologies to Scheherazade - tails of 1001 kinesin motors, Annu. Rev. Genet., 27, 1993, 319–351.[Medline]
Groigno L. Whitaker M.. An anaphase calcium signal controls chromosome disjunction in early sea urchin embryos, Cell, 92, 1998, 193–204.[Medline]
Henson J.H. Cole D.G. Terasaki M. Rashid D. Scholey J.M.. Immunolocalization of the heterotrimeric kinesin-related protein KRP((85/95)) in the mitotic apparatus of sea urchin embryos, Dev. Biol., 171, 1995, 182–194.[Medline]
Hinchcliffe E. Cassels G.O. Rieder C.L. Sluder G.. The coordination of centrosome reproduction with nuclear events of the cell cycle in the sea urchin zygote, J. Cell Biol., 140, 1998, 1417–1426.
Holzbaur E.L.F. Vallee R.B.. Dyneinsmolecular structure and cellular function, Annu. Rev. Cell Biol., 10, 1994, 339–372.[Medline]
Hoyt M.A. Geiser J.R.. Genetic analysis of the mitotic spindle, Annu. Rev. Genet., 30, 1996, 7–33.[Medline]
Karsenti E. Boleti H. Vernos I.. The role of microtubule dependent motors in centrosome movements and spindle pole organization during mitosis, Semin. Cell Dev. Biol., 7, 1996, 367–378.
Kashina A.S. Rogers G.C. Scholey J.M.. The bimC family of kinesinsessential bipolar mitotic motors driving centrosome separation, Biochim. Biophys. Acta., 1357, 1997, 257–271.[Medline]
Kiehart D.P.. Microinjection of echinoderm eggsapparatus and procedures, Methods Cell Biol., 25, 1982, 13–31.[Medline]
Lupus A. Van Dyke M. Stock J.. Predicting coiled coils from protein sequences, Science, 252, 1991, 1162–1164.
Molina I. Baars S. Brill J.A. Hales K.G. Fuller M.T. Ripoll P.. A chromatin-associated kinesin-related protein required for normal mitotic chromosome segregation in Drosophila, J. Cell Biol., 139, 1997, 1361–1371.
Morris R.L. Scholey J.M.. Heterotrimeric kinesin-II is required for the assembly of motile 9+2 cilliary axonemes on sea urchin embryos, J. Cell Biol., 138, 1997, 1009–1022.
Mountain V. Simerly C. Howard L. Ando A. Schatten G. Compton D.A.. The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle, J. Cell Biol., 147, 1999, 351–366.
Nakagawa T. Tanaka Y. Matsuoka E. Kondo S. Okada Y. Noda Y. Kanai Y. Hirokawa N.. Identification and classification of 16 new kinesin superfamily (KIF) proteins in mouse genome, Proc. Natl. Acad. Sci. USA., 94, 1997, 9654–9659.
Nigg E.A.. Cellular substrates of p34cdc2 and its companion cyclin dependent kinases, Trends Cell Biol., 3, 1993, 296–300.[Medline]
O'Connell M.J. Meluh P.B. Rose M.D. Morris N.R.. Suppression of the bimC4 mitotic spindle defect by deletion of klpA, a gene encoding a KAR3-related kinesin-like protein in Aspergillus nidulans, J Cell Biol, 120, 1993, 153–162.
Pidoux A.L. Ledizet M. Cande W.Z.. Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function, Mol. Biol. Cell, 7, 1996, 1639–1655.[Abstract]
Rogers G.C., The functional coordination of three different microtubule-based motors in positioning centrosomes during sea urchin embryogenesis. Ph.D. Thesis, 2000 University of California Davis, CA.
Rogers S. Wells R. Rechsteiner M. Amino acid sequences common to rapidly degraded proteinsthe PEST hypothesis, Science, 234, 1986, 364–368.
Rogers G.C. Hart C.L. Wedaman K.P. Scholey J.M.. Identification of Kinesin-C, a calmodulin-binding carboxy-terminal kinesin in animal (Strongylocentrotus purpuratus) cells, J. Mol. Biol., 294, 1999, 1–8.[Medline]
Saunders W.S. Hoyt M.A.. Kinesin-related proteins required for structural integrity of the mitotic spindle, Cell, 70, 1992, 451–458.[Medline]
Sawin K.E. Leguellec K. Philippe M. Mitchison T.J.. Mitotic spindle organization by a plus-end-directed microtubule motor, Nature, 359, 1992, 540–543.[Medline]
Scholey J.M.. Functions of motor proteins in echinoderm embryosan argument in support of antibody inhibition experiments, Cell Motil. Cytoskel., 39, 1998, 257–260.[Medline]
Sharp D.J. Yu K.R. Sisson J.C. Sullivan W. Scholey J.M.. Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos, Nat. Cell Biol., 1, 1999, 51–54.[Medline]
Sharp D.J. Brown H.M. Kwon M.K. Rogers G.C. Holland G. Scholey J.M.. Functional coordination of three mitotic motors in Drosophila embryos, Mol. Biol. Cell., 11, 2000, 241–253.
Siegel L. Monty K.. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases, Biochim. Biophys. Acta., 112, 1966, 346–362.[Medline]
Song H. Golovkin M. Reddy A.S.N. Endow S.A.. In vitro motility of AtKCPB, a calmodulin-binding kinesin protein of Arabidopsis, Proc. Natl. Acad. Sci. USA., 94, 1997, 322–327.
Starr D.A. Williams B.C. Hays T.S. Goldberg M.L.. ZW10 helps to recruit dynactin and dynein to the kinetochore, J. Cell Biol., 142, 1998, 763–774.
Vaisberg E.A. Koonce M.P. McIntosh J.R.. Cytoplasmic dynein plays a role in mammalian mitotic spindle formation, J. Cell Biol., 123, 1993, 849–858.
Vernos I. Karsenti E.. Chromosomes take the lead in spindle assembly, Trends Cell Biol, 5, 1995, 297–301.[Medline]
Vernos I. Raats J. Hirano T. Heasman J. Karsenti E. Wylie C.. Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning, Cell, 81, 1995, 117–127.[Medline]
Warrick H.M. Spudich J.A.. Myosin structure and function in cell motility, Annu. Rev. Cell Biol., 3, 1987, 379–421.
Wittmann T. Boleti H. Antony C. Karsenti E. Vernos I.. Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubule-associated protein, and dynein, J. Cell Biol., 143, 1998, 673–685.
Wright B.D. Terasaki M. Scholey J.M.. Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell divisionevaluation using antibody microinjection, J. Cell Biol., 123, 1993, 681–689.
Zamyatnin A.A.. Protein volume in solution, Prog. Biophys. Mol. Biol., 24, 1972, 107–123.[Medline]
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