pavarotti


EVOLUTIONARY HOMOLOGS

C. elegans Pavarotti homologs

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. The identification of a null allele of zen-4, an MKLP1 homolog in the nematode Caenorhabditis elegans is reported; ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. It is proposed that ZEN-4 directly cross-links the midzone microtubules and suggested that these microtubules are required for the completion of cytokinesis (Raich, 1998).

Dividing cells need to coordinate the separation of chromosomes with the formation of a cleavage plane. There is evidence that microtubule bundles in the interzone region of the anaphase spindle somehow control both the location and the assembly of the cleavage furrow. A microtubule motor that concentrates in the interzone, MKLP1, has been implicated in the assembly of both the metaphase spindle and the cleavage furrow. To gain insight into mechanisms that might underlie interdependence of the spindle and the cleavage furrow, RNA-mediated interference (RNAi) was used to study the effects of eliminating MKLP1 from Caenorhabditis elegans embryos. Surprisingly, in MKLP1(RNAi) embryos, spindle formation appears normal until late anaphase. Microtubule bundles form in the spindle interzone and the cleavage furrow assembles; anaphase and cleavage furrow ingression initially appear normal. The interzone bundles do not gather into a stable midbody, however, and furrow contraction always fails before complete closure. This sequence of relatively normal mitosis and a late failure of cytokinesis continues for many cell cycles. These and additional results suggest that the interzone microtubule bundles need MKLP1 to encourage the advance and stable closure of the cleavage furrow (Powers, 1998).

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. The cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis (Jantsch-Plunger, 2000).

The Aurora/Ipl1p-related kinase AIR-2 (Drosophila homolog: IplI-aurora-like kinase, also known as Aurora B) is required for mitotic chromosome segregation and cytokinesis in early C. elegans embryos. Previous studies have relied on non-conditional mutations or RNA-mediated interference (RNAi) to inactivate AIR-2. It has therefore not been possible to determine whether AIR-2 functions directly in cytokinesis or if the cleavage defect results indirectly from the failure to segregate DNA. One intriguing hypothesis is that AIR-2 acts to localize the mitotic kinesin-like protein ZEN-4 (also known as CeMKLP1), which later functions in cytokinesis. Using conditional alleles, it has been established that AIR-2 is required at metaphase or early anaphase for normal segregation of chromosomes, localization of ZEN-4, and cytokinesis. ZEN-4 is first required late in cytokinesis, and also functions to maintain cell separation through much of the subsequent interphase. DNA segregation defects alone were not sufficient to disrupt cytokinesis in other mutants, suggesting that AIR-2 acts specifically during cytokinesis through ZEN-4. AIR-2 and ZEN-4 share similar genetic interactions with the formin homology (FH) protein CYK-1, suggesting that AIR-2 and ZEN-4 function in a single pathway, in parallel to a contractile ring pathway that includes CYK-1. Using in vitro co-immunoprecipitation experiments, it has been found that AIR-2 and ZEN-4 interact directly. It is concluded that AIR-2 has two functions during mitosis: one in chromosome segregation, and a second, independent function in cytokinesis through ZEN-4. AIR-2 and ZEN-4 may act in parallel to a second pathway that includes CYK-1 (Severson, 2000).

In all eukaryotic organisms, the physical separation of two nascent cells must be coordinated with chromosome segregation and mitotic exit. In Saccharomyces cerevisiae and Schizosaccharomyces pombe this coordination depends on a number of genes that cooperate in intricate regulatory pathways termed mitotic exit network and septum initiation network, respectively. The function of potentially homologous genes has been explored in a metazoan organism, Caenorhabditis elegans, using RNA-mediated interference. Of all the genes tested, only depletion of CeCDC-14, the C. elegans homolog of the budding yeast dual-specificity phosphatase Cdc14p (Clp1/Flp1p in fission yeast), caused embryonic lethality. CeCDC-14 is required for cytokinesis but may be dispensable for progression of the early embryonic cell cycles. In response to depletion of CeCDC-14, embryos fail to establish a central spindle, and several proteins normally found at this structure are mislocalized. CeCDC-14 itself localizes to the central spindle in anaphase and to the midbody in telophase. It colocalizes with the mitotic kinesin ZEN-4 (Drosophila homolog Pavarotti), and the two proteins depend on each other for correct localization. These findings identify the CDC14 phosphatase as an important regulator of central spindle formation and cytokinesis in a metazoan organism (Gruneberg, 2002).

A late step in cytokinesis requires the central spindle, which forms during anaphase by the bundling of antiparallel nonkinetochore microtubules. Microtubule bundling and completion of cytokinesis in C. elegans requires ZEN-4/CeMKLP-1, a kinesin-like protein, and CYK-4, which contains a RhoGAP domain. CYK-4 and ZEN-4 exist in a complex in vivo that can be reconstituted in vitro. The N terminus of CYK-4 binds the central region of ZEN-4, including the neck linker. Genetic suppression data prove the functional significance of this interaction. An analogous complex, containing equimolar amounts of a CYK-4 ortholog and MKLP-1, has been purified from mammalian cells. Biochemical studies indicate that this complex, named centralspindlin, is a heterotetramer. Centralspindlin, but not its individual components, strongly promotes microtubule bundling in vitro (Mishima, 2002).

Genetic and biochemical suppression of CYK-4(S15L) by mutations in ZEN-4 strongly argues that the interaction between CYK-4 and ZEN-4 is critical for CYK-4 function. Indeed, in vivo, the majority of ZEN-4 is in a complex with CYK-4. Surprisingly, the primary structure of the N-terminal region of CYK-4 is not well conserved. However, the function is likely conserved, since the N-terminal 120 residues of HsCYK-4 are sufficient to localize in cultured mammalian cells. In addition, the ortholog of CYK-4 is required for cytokinesis in mammalian cells and in mouse embryos. Moreover, five pieces of evidence together indicate that HsCYK-4 and MKLP-1 are in a tetrameric complex. (1) Immunopurification of HsCYK-4 and MKLP-1 recovered equimolar amounts of the two proteins. (2) These two proteins comigrate on sucrose density gradients with a similar S value as observed for the C. elegans proteins. (3) The two proteins comigrate on a gel filtration column and their fractionation behavior suggests a native molecular mass for the complex of ~300 kDa. (4) Upon reconstitution of the complex in insect cells, equimolar amounts of CYK-4 copurified with ZEN-4. (5) Both CYK-4 and ZEN-4 are able to individually multimerize. Previous determinations of the native molecular mass of MKLP-1 have been reported, and the values are similar to those presented here. These studies had not taken the presence of CYK-4 into consideration, and therefore the data was interpreted to indicate that MKLP-1 exists as a homotetramer. In contrast, the results of this study indicate that the centralspindlin complex is a tetramer containing two molecules of the ZEN-4/MKLP-1 kinesin and two molecules of the CYK-4 RhoGAP (Mishima, 2002).

CYK-4 binds to ZEN-4 in a particularly interesting domain of this kinesin family member. A critical element of the kinesin molecule lies just C-terminal to the catalytic core -- the neck linker region. ATP binding to one catalytic core induces a large conformational change in the neck linker region that causes the other catalytic core present in the kinesin dimer to extend toward the adjacent tubulin subunit situated on the plus side of the initial microtubule contact. CYK-4 binds to a region of ZEN-4 that includes the neck linker region. In conventional kinesin, the neck linker corresponds to a region 15 amino acids long that connects the catalytic core of kinesin to the coiled-coil stalk domain. Among the family of KIN-N motors, the MKLP-1 subfamily has a distinctly divergent neck linker region; it lacks several nearly invariant residues, and the linker between the catalytic core and the coiled-coil region is about five times longer than in other members of the KIN-N family. The divergence of this critical region of the kinesin suggests that MKLP-1-mediated microtubule motility may differ from that of other kinesins. Moreover, since CYK-4 binds to the neck linker region of ZEN-4, it is possible that CYK-4 binding may in fact regulate ZEN-4 motor activity (Mishima, 2002).

The centralspindlin complex appears to contain two kinesin motors and two RhoGAP molecules. Since most kinesin motors are dimers in which both catalytic cores interact with a single microtubule protofilament, the subunit composition for the complex does not easily explain how microtubule bundling is achieved. If the two kinesin subunits of centralspindlin bind to the same microtubule protofilament, how might microtubule crosslinking occur? At this point, at least three possible, though not mutually exclusive, mechanisms can be invisioned (Mishima, 2002).

The first possibility is that there is an additional microtubule binding site elsewhere in the CYK-4/ZEN-4 complex. No additional binding site has been identified yet in CYK-4, nor have MKLP-1, ZEN-4, or Pav been found to have an additional microtubule binding site. However, it has been shown that MKLP-1 interacts differently with microtubules than it does with most kinesin-like proteins. Specifically, ATP is usually sufficient to elute most kinesins from microtubules, but in the case of MKLP-1, both ATP and high salt are required (Kuriyama, 1994; Nislow, 1992). Thus it is possible that MKLP-1 interacts with two microtubules, one by the motor domain and another via a different interaction surface. Consistent with this possibility is the finding that Rab6KIFL kinesin, which is quite similar to MKLP-1 in primary structure as well as in its localization and proposed function, contains a second microtubule binding activity in the C-terminal half of the molecule. However, this possibility does not explain why CYK-4 is required for central spindle assembly (Mishima, 2002 and references therein).

The second alternative is that MKLP-1 forms higher order structures than that of the tetramer. This possibility gains some support from the biochemical characterization of centralspindlin. In vitro, centralspindlin forms higher order complexes at physiological ionic strength. Further support of this possibility comes from localization studies; in both C. elegans and mammalian cells, ring-like structures are found that have been termed division remnants. These persist in the cell cortex after division. These remnants appear to be large aggregates of centralspindlin, which are not in obvious association with microtubule bundles. Higher order oligomers could potentially form in early anaphase and promote microtubule bundling. This model is conceptually similar to the mechanism by which myosin II filaments promote the formation of antiparallel bundles of actin filaments (Mishima, 2002).

The third possibility is that, unlike most N-terminal kinesins, the two catalytic cores of the kinesin subunits in the centralspindlin complex could bind to different microtubules. This would not be without precedent, in that the KIN-N KIF1A moves processively along a microtubule using a single head. The association of the two catalytic cores of MKLP-1 with different microtubules is made feasible by the fact that the linker region between the catalytic core and the coiled-coil domain is much longer than that present in most N-terminal kinesins. Perhaps CYK-4 ensures that the two motor domains are oriented in such a way to bind to antiparallel microtubules. Structural analysis of centralspindlin will help to define the mechanism of antiparallel microtubule bundling (Mishima, 2002).

Several lines of evidence suggest that centralspindlin may be regulated by at least two different kinases. Genetic analysis indicates that Pav localization requires Polo kinase. In addition, the Aurora-B/Incenp complex is also required for the stable localization of centralspindlin in C. elegans embryos, and an in vitro biochemical interaction has been detected between Aurora-B (AIR-2) and ZEN-4. Reconstitution of central spindle assembly in vitro will allow the role of these and other regulators of centralspindlin to be dissected (Mishima, 2002 and references therein).

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). This study describes a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability (Mishima, 2004).

Epithelial tubes are a key component of organs and are generated from cells with distinct apico-basolateral polarity. A novel function during tubulogenesis is described for ZEN-4, the Caenorhabditis elegans ortholog of mitotic kinesin-like protein 1 (MKLP1), and CYK-4, which contains a RhoGAP (GTPase-activating protein) domain. Previous studies have revealed that these proteins comprise centralspindlin (a complex that functions during mitosis to bundle microtubules), construct the spindle midzone, and complete cytokinesis. ZEN-4/MKLP1 functions postmitotically to establish the foregut epithelium. Mutants that lack ZEN-4/MKLP1 express polarity markers but fail to target these proteins appropriately to the cell cortex. Affected proteins include PAR-3/Bazooka and PKC-3/atypical protein kinase C at the apical membrane domain, and HMR-1/cadherin and AJM-1 within C. elegans apical junctions (CeAJ). Microtubules and actin are disorganized in zen-4 mutants compared to the wild-type. It is suggested that ZEN-4/MKLP1 and CYK-4/RhoGAP regulate an early step in epithelial polarization that is required to establish the apical domain and CeAJ (Portereiko, 2004).

Identification and mitotic functions of vertebrate Pavarotti homologs

A monoclonal antibody raised against mitotic spindles isolated from CHO cells (CHO1) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase (Nislow, 1990).

Mitosis comprises a complex set of overlapping motile events, many of which involve microtubule-dependent motor enzymes. A new member of the kinesin superfamily is described. The protein was originally identified as a spindle antigen by the CHO1 monoclonal antibody and shown to be required for mitotic progression. The gene that encodes this antigen has been cloned; the encoded sequence contains a domain with strong sequence similarity to the motor domain of kinesin-like proteins. The product of this gene, expressed in bacteria, can cross-bridge antiparallel microtubules in vitro, and in the presence of Mg-ATP, microtubules slide over one another in a fashion reminiscent of microtubule movements during spindle elongation (Nislow, 1992).

The CHO1 antigen is a mitosis-specific kinesin-like motor located at the interzonal region of the spindle. The human cDNA coding for the antigen contains a domain with sequence similarity to the motor domain of kinesin-like protein. cDNAs encoding the CHO1 antigen have been cloned by immunoscreening of a CHO Uni-Zap expression library, the same species in which the original monoclonal antibody was raised. cDNAs of CHO cells encode a 953 amino acid polypeptide with a calculated molecular mass of 109 kDa. The N-terminal 73% of the antigen was 87% identical to the human clone, whereas the remaining 27% of the coding region showed only 48% homology. Insect Sf9 cells infected with baculovirus containing the full-length insert produced 105 and 95 kDa polypeptides, the same doublet identified as the original antigen in CHO cells. Truncated polypeptides corresponding to the N-terminal motor and C-terminal tail produced a 56 and 54 kDa polypeptide in Sf9 cells, respectively. Full and N-terminal proteins co-sedimented with, and caused bundling of, brain microtubules in vitro, whereas the C-terminal polypeptide did not. Cells expressing the N terminus formed one or more cytoplasmic processes. Immunofluorescence as well as electron microscopic observations have revealed the presence of thick bundles of microtubules, which are closely packed, forming a marginal ring just beneath the cell membrane and a core in the processes. The diffusion coefficient and sedimentation coefficient were determined for the native CHO1 antigen by gel filtration and sucrose density gradient centrifugation, respectively. The native molecular mass of overinduced protein in Sf9 cells was calculated as 219 kDa, suggesting that the antigen exists as a dimer. Intrinsic CHO1 antigen in cultured mammalian cells forms a larger native complex (native molecular mass, 362 kDa), which may suggest the presence of additional molecule(s) associating with the CHO1 motor molecule (Kuriyama, 1994).

To understand the functions of microtubule motors in vertebrate development, the kinesin-like proteins (KLPs) of the zebrafish, Danio rerio, were investigated. This study describes the structure, intracellular distribution, and function of zebrafish mitotic KLP1 (Mklp1). The zebrafish mklp1 gene that encodes this 867-amino acid protein maps to a region of zebrafish linkage group 18 that is syntenic with part of human chromosome 15. In zebrafish AB9 fibroblasts and in COS-7 cells, the zebrafish Mklp1 protein decorates spindle microtubules at metaphase, redistributes to the spindle midzone during anaphase, and becomes concentrated in the midbody during telophase and cytokinesis. The motor is detected consistently in interphase nuclei of COS cells and occasionally in those of AB9 cells. Nuclear targeting of Mklp1 is conferred by two basic motifs located in the COOH terminus of the motor. In cleaving zebrafish embryos, green fluorescent protein (GFP)-tagged Mklp1 is found in the nucleus in interphase and associates with microtubules of the spindle midbody in cytokinesis. One- or two-cell embryos injected with synthetic mRNAs encoding dominant-negative variants of GFP-Mklp1 frequently fail to complete cytokinesis during cleavage, resulting in formation of multinucleated blastomeres. These results indicate that the zebrafish Mklp1 motor performs a critical function that is required for completion of embryonic cytokinesis (Chen, 2000).

CHO1 is a mammalian kinesin-like motor protein of the MKLP1 subfamily. It associates with the spindle midzone during anaphase and concentrates to a midbody matrix during cytokinesis. CHO1 was originally implicated in karyokinesis, but the invertebrate homologs of CHO1 were shown to function in the midzone formation and cytokinesis. To analyze the role of the protein in mammalian cells, the ATP-binding site of CHO1 was mutated and expressed in CHO cells. Mutant protein (CHO1F') is able to interact with microtubules via ATP-independent microtubule-binding site(s) but fails to accumulate at the midline of the central spindle and affects the localization of endogenous CHO1. Although the segregation of chromosomes, the bundling of midzone microtubules, and the initiation of cytokinesis proceeds normally in CHO1F'-expressing cells, the completion of cytokinesis is inhibited. Daughter cells frequently enter interphase while connected by a microtubule-containing cytoplasmic bridge from which the dense midbody matrix is missing. Depletion of endogenous CHO1 via RNA-mediated interference also affects the formation of midbody matrix in dividing cells, causes the disorganization of midzone microtubules, and results in abortive cytokinesis. Thus, CHO1 may not be required for karyokinesis, but it is essential for the proper midzone/midbody formation and cytokinesis in mammalian cells (Matuliene, 2002).

CHO1 is a kinesin-like protein of the mitotic kinesin-like protein (MKLP)1 subfamily present in central spindles and midbodies in mammalian cells. It is different from other subfamily members in that it contains an extra approximately 300 bp in the COOH-terminal tail. Analysis of the chicken genomic sequence shows that heterogeneity is derived from alternative splicing, and exon 18 is expressed in only the CHO1 isoform. CHO1 and its truncated isoform MKLP1 are coexpressed in a single cell. Surprisingly, the sequence encoded by exon 18 possesses a capability to interact with F-actin, suggesting that CHO1 can associate with both microtubule and actin cytoskeletons. Microinjection of exon 18-specific antibodies did not result in any inhibitory effects on karyokinesis and early stages of cytokinesis. However, almost completely separated daughter cells became reunited to form a binulceate cell, suggesting that the exon 18 protein may not have a role in the formation and ingression of the contractile ring in the cortex. Rather, it might be involved directly or indirectly in the membrane events necessary for completion of the terminal phase of cytokinesis (Kuriyama, 2002).

CHO1 is a mammalian kinesin-like motor protein of the MKLP1 subfamily. It associates with the spindle midzone during anaphase and concentrates to a midbody matrix during cytokinesis. CHO1 was originally implicated in karyokinesis, but the invertebrate homologs of CHO1 were shown to function in the midzone formation and cytokinesis. To analyze the role of the protein in mammalian cells, the ATP-binding site of CHO1 was mutated and expressed in CHO cells. Mutant protein (CHO1F') is able to interact with microtubules via ATP-independent microtubule-binding site(s) but fails to accumulate at the midline of the central spindle and affects the localization of endogenous CHO1. Although the segregation of chromosomes, the bundling of midzone microtubules, and the initiation of cytokinesis proceeds normally in CHO1F'-expressing cells, the completion of cytokinesis is inhibited. Daughter cells frequently enter interphase while connected by a microtubule-containing cytoplasmic bridge from which the dense midbody matrix is missing. Depletion of endogenous CHO1 via RNA-mediated interference also affects the formation of midbody matrix in dividing cells, causes the disorganization of midzone microtubules, and results in abortive cytokinesis. Thus, CHO1 may not be required for karyokinesis, but it is essential for the proper midzone/midbody formation and cytokinesis in mammalian cells (Matuliene, 2002).

Nuclear localization of C-terminal domains of the kinesin-like protein MKLP-1

The successful execution of mitosis in mammalian cells requires the activities of numerous kinesin-like proteins. The Mitotic Kinesin-Like Protein-1 (MKLP-1) localizes to the spindle equator and is believed to participate in the separation of spindle poles during anaphase B. Injection of antibodies against MKLP-1 into dividing cells results in cell cycle arrest, suggesting that MKLP-1 is essential for mitosis. To further characterize MKLP-1, constructs encoding C-terminal domains of MKLP-1 were expressed as fusions to the green fluorescent protein and localized in HeLa cells. All constructs localized to the nucleus indicating the presence of at least one nuclear localization sequence in the C-terminus of the protein. C-terminal domains of MKLP-1 expressed in insect cells also localized to the nucleus as shown by subcellular fractionation. These proteins remained tightly associated with the nucleus following both detergent and salt extraction, suggesting a tight interaction with a component of the nucleus (Deavpirs, 1999).

Interaction of Pavarotti homologs with Polo kinase and with phosphatases

PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions. An affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts. The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells. Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells. Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis. Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity. Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis. Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase. In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro (Lee, 1995).

Interaction of Pavarotti homologs with Arf proteins

Arf proteins comprise a family of 21-kDa GTP-binding proteins with many proposed functions in mammalian cells, including the regulation of several steps of membrane transport, maintenance of organelle integrity, and activation of phospholipase D. A yeast two-hybrid screen of human cDNA libraries was performed using a dominant activating allele, [Q71L], of human Arf3 as bait. Eleven independent isolates contained plasmids encoding the C-terminal tail of mitotic kinesin-like protein-1 (MKLP1). Further deletion mapping allowed the identification of an 88 amino acid Arf3 binding domain in the C-terminus of MKLP1. This domain has no clear homology to other Arf binding proteins or to other proteins in the protein databases. The C-terminal domain of MKLP1 was expressed and purified from bacteria as a GST fusion protein and shown to bind Arf3 in a GTP-dependent fashion. A screen for mutations in Arf3 that specifically lost the ability to bind MKLP1 identified 10 of 14 point mutations in the GTP-sensitive switch I or switch II regions of Arf3. Two-hybrid assays of the C-terminal domain of MKLP1 with each of the human Arf isoforms revealed strong interaction with each. Taken together, these data are all supportive of the conclusion that activated Arf proteins bind to the C-terminal 'tail' domain of MKLP1 (Boman, 1999).

Phosphorylation of ZEN-4/MKLP1 by aurora B regulates completion of cytokinesis

The central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules, and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase (see Drosophila Aurora B)and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly. Centralspindlin localization depends on aurora B kinase. Aurora B concentrates in the midbody and persists between daughter cells. In C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis (Guse, 2005).

Expression and function of Pavarotti homologs in post-mitotic neurons

The microtubules (MTs) within neuronal processes are highly organized with regard to their polarity and yet are not attached to any detectable nucleating structure. Axonal MTs are uniformly oriented with their plus ends distal to the cell body, whereas dendritic MTs are of both orientations. The capacity of motor-driven MT transport to organize distinct MT patterns during process outgrowth has been tested. Focus was placed on CHO1/MKLP1, a kinesin-related protein present in the midzonal region of the mitotic spindle where MTs of opposite orientation overlap. Insect ovarian Sf9 cells induced to express the N-terminal portion of the molecule form MT-rich processes with a morphology similar to that of neuronal dendrites. Nascent processes contain uniformly plus-end-distal MTs, but these are joined by minus-end-distal MTs as the processes continue to develop. Thus, this CHO1/MKLP1 fragment establishes a nonuniform MT polarity pattern and does so by a similar sequence of events as occurs with the dendrite, the antecedent of which is a short process with a uniform MT polarity orientation. Two lines of evidence suggest that these results are elicited by motor-driven MT transport: (1) there is a depletion of MTs from the cell body during process outgrowth; (2) the same polarity pattern is obtained when net MT assembly is suppressed pharmacologically during process formation. Collectively, these findings provide precedent for the idea that motor-driven transport can organize MTs into distinct patterns of polarity orientation during process outgrowth (Sharp, 1996).

The quintessential feature of the dendritic microtubule array is its nonuniform pattern of polarity orientation. During the development of the dendrite, a population of plus end-distal microtubules first appears, and these microtubules are subsequently joined by a population of oppositely oriented microtubules. The latter microtubules are intercalated within the microtubule array by their specific transport from the cell body of the neuron during a critical stage in development. In addition, the mitotic motor protein termed CHO1/MKLP1 has the appropriate properties to transport microtubules in this manner. This study attempts to determine whether CHO1/MKLP1 continues to be expressed in terminally postmitotic neurons and whether it is required for the establishment of the dendritic microtubule array. In situ hybridization analyses reveal that CHO1/MKLP1 is expressed in postmitotic cultured rat sympathetic and hippocampal neurons. Immunofluorescence analyses indicate that the motor is absent from axons but is enriched in developing dendrites, where it appears as discrete patches associated with the microtubule array. Treatment of the neurons with antisense oligonucleotides to CHO1/MKLP1 suppresses dendritic differentiation, presumably by inhibiting the establishment of their nonuniform microtubule polarity pattern. It is concluded that CHO1/MKLP1 transports microtubules from the cell body into the developing dendrite with their minus ends leading, thereby establishing the nonuniform microtubule polarity pattern of the dendrite (Sharp, 1997).

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. These distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, studies were performed on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. It is speculated that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern (Yu, 1997).

The kinesin-related motor protein CHO1/MKLP1 was initially thought to be expressed only in mitotic cells, where it presumably transports oppositely oriented microtubules relative to one another in the spindle mid-zone. CHO1/MKLP1 is also expressed in cultured neuronal cells, where it is enriched in developing dendrites. The putative function of CHO1/MKLP1 in these postmitotic cells is to intercalate minus-end-distal microtubules among oppositely oriented microtubules within developing dendrites, thereby establishing their non-uniform microtubule polarity pattern. In situ hybridization has been used to determine whether CHO1/MKLP1 is expressed in a variety of rodent neurons both in vivo and in vitro. These analyses reveal that CHO1/MKLP1 is expressed within various neuronal populations of the brain including those in the cerebral cortex, hippocampus, olfactory bulb and cerebellum. The messenger ribonucleic acid (mRNA) levels are high within these neurons well after the completion of their terminal mitotic division and throughout the development of their dendrites. After this, the levels decrease and are relatively low within the adult brain. Parallel analyses on developing hippocampal neurons in culture indicate that the levels of expression increase dramatically just prior to dendritic development, and then decrease somewhat after the dendrites have differentiated. Dorsal root ganglion neurons, which generate axons but not dendrites, express significantly lower levels of mRNA for CHO1/MKLP1 than hippocampal or sympathetic neurons. These results are consistent with the proposed role of CHO1/MKLP1 in establishing the dendritic microtubule array (Ferhat, 1998).

Dendrites are short stout tapering processes that are rich in ribosomes and Golgi elements, whereas axons are long thin processes of uniform diameter that are deficient in these organelles. It has been hypothesized that the unique morphological and compositional features of axons and dendrites result from their distinct patterns of microtubule polarity orientation. The microtubules within axons are uniformly oriented with their plus ends distal to the cell body, whereas microtubules within dendrites are nonuniformly oriented. The minus-end-distal microtubules are thought to arise via their specific transport into dendrites by the motor protein known as CHO1/MKLP1. According to this model, CHO1/MKLP1 transports microtubules with their minus ends leading into dendrites by generating forces against the plus-end-distal microtubules, thus creating drag on the plus-end-distal microtubules. Depletion of CHO1/MKLP1 from cultured neurons causes a rapid redistribution of microtubules within dendrites such that minus-end-distal microtubules are chased back to the cell body while plus-end-distal microtubules are redistributed forward. The dendrite grows significantly longer and thinner, loses its taper, and acquires a progressively more axon-like organelle composition. These results suggest that the forces generated by CHO1/MKLP1 are necessary for maintaining the minus-end-distal microtubules in the dendrite, for antagonizing the anterograde transport of the plus-end-distal microtubules, and for sustaining a pattern of microtubule organization necessary for the maintenance of dendritic morphology and composition. It is concluded that dendritic identity is dependent on forces generated by CHO1/MKLP1 (Yu, 2000).

Expression and function of Pavarotti homologs in kidney cells

Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood. The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes, the functional significance of the microtubular system in major processes was studied. The following data were obtained: (1) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration; (2) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes; (3) treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs; (4) during the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments are distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (5) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation (Kobayashi, 1998).

The vertebrate-specific Kinesin-6, Kif20b, is required for normal cytokinesis of polarized cortical stem cells and cerebral cortex size

Mammalian neuroepithelial stem cells divide using a poorly understood polarized form of cytokinesis. The cytokinetic furrow cleaves the cell by ingressing from basal to apical, forming the midbody at the apical membrane. The midbody mediates abscission by recruiting many factors, including the Kinesin-6 family member Kif20b. In developing embryos, Kif20b mRNA is most highly expressed in neural stem/progenitor cells. A loss-of-function mutant in Kif20b, magoo, was found in a forward genetic screen. magoo has a small cerebral cortex, with reduced production of progenitors and neurons, but preserved layering. In contrast to other microcephalic mouse mutants, mitosis and cleavage furrows of cortical stem cells appear normal in magoo. However, apical midbodies show changes in number, shape and positioning relative to the apical membrane. Interestingly, the disruption of abscission does not appear to result in binucleate cells, but in apoptosis. Thus, Kif20b is required for proper midbody organization and abscission in polarized cortical stem cells and has a crucial role in the regulation of cerebral cortex growth (Janisch, 2013).

The spatial and temporal regulation of abscission influence the partitioning of cytoplasm and membrane between daughters, but also determine inheritance of the midbody. In some cells, abscission occurs on one side of the midbody, so one daughter inherits it. Other studies have observed abscission on both sides of the midbody. Intriguingly, midbody inheritance was recently linked to cell fate. One group showed that embryonic stem cells and cancer cells tend to accumulate midbodies. Another group reported that neural stem cell divisions release midbodies with two abscissions, and hypothesized that this is important to maintain symmetric daughter fates. Loss of Kif20b could disrupt double abscissions or the control of midbody inheritance in neural stem cells. Much more work is needed to understand the roles of abscission and midbody inheritance in cell fate (Janisch, 2013).

In conclusion, future insights into development and disease will require a better understanding of cytokinesis mechanisms in different cell types, complex tissues and symmetric versus asymmetric divisions. The Kif20bmagoo mutant offers a new model of microcephaly, different from previous mouse models, that shows abnormalities specific to the mitosis of cortical progenitors. This and other work suggest that the regulation of cytokinetic abscission, in addition to the regulation of mitosis and cleavage, could play a previously unappreciated role in stem cell renewal and the control of cerebral cortex size (Janisch, 2013).

SHCBP1 is required for midbody organization and cytokinesis completion

The centralspindlin complex, which is composed of MKLP1 and MgcRacGAP, is one of the crucial factors involved in cytokinesis initiation. Centralspindlin is localized at the middle of the central spindle during anaphase and then concentrates at the midbody to control abscission. A number of proteins that associate with centralspindlin have been identified. These associating factors regulate furrowing and abscission in coordination with centralspindlin. A recent study identified a novel centralspindlin partner, called Nessun Dorma, which is essential for germ cell cytokinesis in Drosophila melanogaster. SHCBP1 is a human ortholog of Nessun Dorma that associates with human centralspindlin. This report analyzes the interaction of SHCBP1 with centralspindlin in detail and determined the regions that are required for the interaction. In addition, it was demonstrated that the central region is necessary for the SHCBP1 dimerization. Both MgcRacGAP and MKLP1 are degraded once cells exit mitosis. Similarly, endogenous and exogenous SHCBP1 were degraded with mitosis progression. Interestingly, SHCBP1 expression was significantly reduced in the absence of centralspindlin, whereas centralspindlin expression was not affected by SHCBP1 knockdown. Finally, it was demonstrated that SHCBP1 depletion promotes midbody structure disruption and inhibits abscission, a final stage of cytokinesis. This study gives novel insight into the role of SHCBP in cytokinesis completion (Asano, 2014).


pavarotti: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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