Kinesin-like Protein Research Articles

Plk Is an M-Phase-Specific Protein Kinase and Interacts with a Kinesin-Like Protein, CHO1/MKLP-1

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. 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.

Identification of the chromosome localization domain of the Drosophila nod kinesin-like protein.

The nod kinesin-like protein is localized along the arms of meiotic chromosomes and is required to maintain the position of achiasmate chromosomes on the developing meiotic spindle. Here we show that the localization of ectopically expressed nod protein on mitotic chromosomes precisely parallels that observed for wild-type nod protein on meiotic chromosomes. Moreover, the carboxyl-terminal half of the nod protein also binds to chromosomes when overexpressed in mitotic cells, whereas the overexpressed amino-terminal motor domain binds only to microtubules. Chromosome localization of the carboxyl-terminal domain of nod depends upon an 82-amino acid region comprised of three copies of a sequence homologous to the DNA-binding domain of HMG 14/17 proteins. These data map the two primary functional domains of the nod protein in vivo and provide a molecular explanation for the directing of the nod protein to a specific subcellular component, the chromosome.

Motor activity and mitotic spindle localization of the Drosophila kinesin-like protein KLP61F.

The KLP61F gene product is essential for Drosophila development. Mutations in KLP61F display a mitotic arrest phenotype caused by a failure in the proper separation of duplicated centrosomes (Heck et al., 1993). Sequence analysis of KLP61F identified it as a member of the bimC family of kinesin-like microtubule motor proteins. Here we report that KLP61F is distinct from KRP130, a kinesin-like protein recently purified from Drosophila embryos and suggested to be the product of the KLP61F gene (Cole et al., 1994). We also characterized recombinant KLP61F and found that it possesses microtubule-stimulated ATPase and microtubule translocation activities in vitro. In addition, we have used an affinity-purified, KLP61F-specific antiserum to localize native KLP61F and an epitope-tagged KLP61F fusion protein during various stages of mitosis in Drosophila syncytial blastoderm embryos. From early prophase through anaphase, KLP61F is coincident with the distribution of tubulin. Together these results confirm the existence of multiple bimC-like kinesins in Drosophila and suggest that KLP61F function is intrinsic to the mitotic spindle.

Effect of hypoxia on human cytotrophoblast differentiation, in vitro

Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins present in exosomes secreted by tumor cells have been poorly defined. This study describes the protein composition of mesothelioma cell-derived exosomes in more detail. After electrophoresis of exosome preparations, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to characterize the protein spots. MHC class I was found to be present together with the heat shock proteins HSC70 and HSP90. In addition, we found annexins and PV-1, proteins involved in membrane transport and function. Cytoskeleton proteins and their associated proteins ezrin, moesin, actinin-4, desmoplakin, and fascin were also detected. Besides the molecular motor kinesin-like protein, many enzymes were detected revealing the cytoplasmic orientation of exosomes. Most interesting was the detection of developmental endothelial locus-1 (DEL-1), which can act as a strong angiogenic factor and can increase the vascular development in the neighborhood of the tumor. In conclusion, mesothelioma cells release exosomes that express a discrete set of proteins involved in antigen presentation, signal transduction, migration, and adhesion. Exosomes may play an important role in the interaction between tumor cells and their environment.

Chromosomes take the lead in spindle assembly

Several kinesin-like motor proteins have recently been found associated with chromosome arms. They seem to be involved in the so-called ‘polar ejection forces’ that contribute to the congression of chromosomes on the metaphase plate, and at least one of them is essential for the maintenance of spindle bipolarity. The discovery of these molecules changes our view of the mechanism of spindle assembly and chromosome movement.

Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.

The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis.

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.

DNA binding and meiotic chromosomal localization of the drosophila nod kinesin-like protein

The Drosophila no distributive disjunction (nod) gene encodes a kinesin-like protein that has been proposed to push chromosomes toward the metaphase plate during female meiosis. We report that the nonmotor domain of the nod protein can mediate direct binding to DNA. Using an antiserum prepared against bacterially expressed nod protein, we show that during prometaphase nod protein is localized on oocyte chromosomes and is not restricted to either specific chromosomal regions or to the kinetochore. Thus, motorbased chromosome-microtubule interactions are not limited to the centromere, but extend along the chromosome arms, providing a molecular explanation for the polar ejection force.

Xklp15 a chromosomal xenopus kinesin-like protein essential for spindle organization and chromosome positioning

Xklp1 is a novel Xenopus kinesin-like protein with a motor domain at the amino terminus, nuclear localization sequences in the stalk, and a putative zinc fingerlike sequence in the tail. It is nuclear during interphase and chromosomal during mitosis. During late anaphase, a fraction of the protein relocalizes to the spindle interzone and accumulates in the midbody during telophase. Depletion of Xklp1 protein by antisense oligo knockout in oocytes leads to defective mitosis during the first cell cycles following fertilization. The bipolarity of spindles assembled in vitro in the presence of anti-Xklpl antibodies is unstable, and the chromosomes fail to congress on the metaphase plate.

Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro.

Chromosomes can move with the ends of depolymerizing microtubules (MTs) in vitro, even in the absence of nucleotide triphosphates (Coue, M., V. A. Lombillo, and J. R. McIntosh. 1991. J. Cell Biol. 112:1165-1175.) Here, we describe an immunological investigation of the proteins important for this form of motility. Affinity-purified polyclonal antibodies to kinesin exert a severe inhibitory effect on depolymerization-dependent chromosome motion. These antibodies predominantly recognize a polypeptide of M(r) approximately 250 kD on immunoblots of CHO chromosomes and stain kinetochores as well as some vesicles that are in the chromosome preparation. Antibodies to CENP-E, a kinetochore-associated kinesin-like protein, also recognize a 250-kD electrophoretic component, but they stain only the kinetochroe region of isolated chromosomes. Polyclonal antibodies that recognize specific domains of the CENP-E polypeptide affect MT disassembly-dependent chromosome motion in different ways; antibodies to the head or tail portions slow motility threefold, while those raised against the neck region stop motion completely. Analogous antibodies that block conventional, ATP-dependent motility of cytoplasmic dynein (Vaisberg, G., M. P. Koonce, and J. R. McIntosh. 1993. J. Cell Biol. 123:849-858) have no effect on disassembly-dependent chromosome motion, even though they bind to kinetochores. These observations suggest that CENP-E helps couple chromosomes to depolymerizing MTs. A similar coupling activity may allow spindle MTs to remain kinetochore-bound while their lengths change during both prometaphase and anaphase A.

CDNA structure and characterization of a kinesin‐like protein from the silkworm Bombyx mori

We have isolated a 1224 bp cDNA clone from a Bombyx mori embryonic cDNA library which contains sequences homologous to the kinesin-like protein gene, ncd, which is required for distribution of chromosomes at meiosis in Drosophila melanogaster females. This clone includes both a microtubule motor and the ATP-binding domains found in kinesin-like proteins. The motor domain is classified in the group of the BimC and cut7, which have a role in spindle formation during mitosis of Aspergillus nidulans and Schizosaccharomyces pombe, respectively. However, the location of the domain at the carboxy terminus is not common in this family, except for ncd and KAR3.

Mitotic regulation of microtubule cross-linking activity of CENP-E kinetochore protein.

CENP-E is a kinesin-like protein that is transiently bound to kinetochores during early mitosis, becomes redistributed to the spindle midzone at anaphase, and is degraded after cytokinesis. At anaphase, CENP-E may cross-link the interdigitating microtubules in the spindle midzone through a motor-like binding site at the amino terminus and a 99-amino acid carboxyl-terminal domain that bound microtubules in a distinct manner. Phosphorylation of the carboxyl terminus by the mitotic kinase maturation promoting factor (MPF) inhibited microtubule-binding activity before anaphase. Thus, MPF suppresses the microtubule cross-linking activity of CENP-E until anaphase, when its activity is lost.

Kinesin-like proteins in the flagella of Chlamydomonas

The flagella of the biflagellate unicellular alga Chlamydomonas have long been known to contain the microtubule-dependent motor protein dynein, but recent findings indicate they also contain multiple members of the kinesin superfamily. Two of these kinesin-like proteins are restricted to a single central-pair microtubule, raising the question of how proteins are targeted to specific microtubules within the flagellum. The kinesin-like proteins on the central-pair microtubules may cause the central-pair apparatus to rotate or twist during flagellar beating. Other kinesins within the flagellum may participate in movements associated with the flagellar membrane.

Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis.

CENP-E is a kinesin-like protein that binds to kinetochores through the early stages of mitosis, but after initiation of anaphase, it relocalizes to the overlapping microtubules in the midzone, ultimately concentration in the developing midbody. By immunoblotting of cells separated at various positions in the cell cycle using centrifugal elutriation, we show that CENP-E levels increase progressively across the cycle peaking at approximately 22,000 molecules/cell early in mitosis, followed by an abrupt (> 10 fold) loss at the end of mitosis. Pulse-labeling with [35S]methionine reveals that beyond a twofold increase in synthesis between G1 and G2, interphase accumulation results primarily from stabilization of CENP-E during S and G2. Despite localizing in the midbody during normal cell division, CENP-E loss at the end of mitosis is independent of cytokinesis, since complete blockage of division with cytochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degradation of as yet unidentified components at the metaphase/anaphase transition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of timed proteolysis.

Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface.

Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.

A structure-function analysis of NOD, a kinesin-like protein from Drosophila melanogaster.

We have analyzed a collection of 12 mutations in the Drosophila melanogaster nod locus, which encodes a kinesin-like protein involved in female meiotic chromosome segregation. The kinesin-like domain is at the N-terminus of the protein, while the C-terminal portion of the protein is unique. Four of the mutations are missense and affect highly conserved domains of the kinesin-like portion of the predicted protein, and thus demonstrate that the sequence conservation is biologically relevant. Surprisingly, two other mutations, which behave genetically as null alleles, are the result of mutations in the last exon of the nod gene. Thus, these two mutations affect the most C-terminal residues in the unique portion of the predicted protein. Based on these mutations, we suggest that this part of the protein may also be essential for wild-type function. The mutations were induced by either gamma-rays or ethyl methanesulfonate (EMS). All of the gamma-ray induced mutations were small or large chromosomal rearrangements, while all of the EMS mutations were G-->A transitions. These findings are consistent with the biochemical basis of the mode of action of each mutagen.

Filamentous fungi: old mutants and new discoveries

Recently, a group of scientists from the fungal genetics community gathered for the biennial EMBO Workshop on the Molecular Biology of Filamentous Fungi (18-21 September, 1993, Mallorca; organized by M.A. Pefialva and C. Scazzocchio). This year, the assembly included some who might be considered outsiders, in the sense that their organism of choice is not a filamentous fungus. However as similarities between systems emerged for example, it was revealed that the conidiation gene abaA from Aspergillus nidulans induces pseudohyphal growth in Saccharomyces cerevisiaeit became obvious that these outsiders were to be dragged into the fold. Since the eady days of fungal genetics when the 'one gene, one protein' hypothesis was defined using auxotrophic mutants of Neurospora crass#, it has often been asserted that fungi are paradigm organisms for the primary investigation of fundamental processes of genetics and cell biology, The validity of this assertion was reinforced in the opening lecture by R. Morris (New Jersey), who presented insights into the cell cycle of A. nMulans. A series of longestablished mutants formed the basis of later molecular research, and conditional lethal mutants with defects in mitosis originally isolated some 20 years ago have now been characterized using molecular techniques. For example, we now know that gene nimA encodes a protein kinase, nimE a B-type cyclin, bimC a kinesin-like protein, bimD a putative DNA-binding protein, bimG a phosphoprotein phosphatase and nudC a protein involved in nuclear migration. Similarly, a 30-year-old mutation in the A mating-type factor of the basidiomycete Coprinus cinereus that causes self-compatibility is now known to result from fusion of two genes that encode two different types of homeodomain transcription factors (L.A. Casselton, Oxford). These homeodomain proteins are related to those of the b mating-type locus of Ustilago maydis and to the a l and a2 proteins, which regulate sexual development in S. cerevisiae. In contrast to yeast, which has only these two homeodomain mating-type proteins, C. cinereus, which has multiple mating types, has a large number of functionally equivalent proteins. R. Kahmann (Munich) reported on the second mating-type locus of U. maydis, the a locus, which encodes a pair of pheromones and their respective pheromone receptors. Genes belonging to both U. maydis mating-type loci are upregulated in response to these pheromones. Now the hunt is on for the genes involved in this signal transduction pathway and the target genes regulated by the bgenes. Interactions between regulatory proteins and structural genes were described in conidiogenesis in A. nidulan~. genes such as brlA, abaA and wetA (which encode transcription factors), rodA (hydrophobin gene) and yA (encodes laccase). The zinc finger protein BrlA and AbaA, a protein with a TEA DNA-binding motif 2, control expression of structural genes inw~lved in conidiation and also regulate each other's expression in a reciprocal manner, abaA and wetA operate via a feedback control, brlA produces two transcripts, one of which is constitutive but subject to a translational check. These two transcripts are important in temporal regulation of development (W. Timberlake, Athens, USA). The instability of transforming DNA sequences during sexual crosses led to the discoveries of the repeat-induced point mutation, RIP, in N. crassa (E. Selker, Oregon) and methylation induced premeiotically, MIP, in Ascobolus immersus (G. Faugeron, Paris). These premeiotic phenomena are similar in that both recognize linked and unlinked duplications and cause the DNAs to become cytosine methylated de novo. However, DNA sequences that have undergone RIP usually have GC-*AT transitions, while the sole

Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell division: evaluation using antibody microinjection.

Previous studies suggest that kinesin heavy chain (KHC) is associated with ER-derived membranes that accumulate in the mitotic apparatus in cells of early sea urchin embryos (Wright, B. D., J. H. Henson, K. P. Wedaman, P. J. Willy, J. N. Morand, and J. M. Scholey. 1991. J. Cell Biol. 113:817-833). Here, we report that the microinjection of KHC-specific antibodies into these cells has no effect on mitosis or ER membrane organization, even though one such antibody, SUK4, blocks kinesin-driven motility in vitro and in mammalian cells. Microinjected SUK4 was localized to early mitotic figures, suggesting that it is able to access kinesin in spindles. In contrast to KHC-specific antibodies, two antibodies that react with kinesin-like proteins (KLPs), namely CHO1 and HD, disrupted mitosis and prevented subsequent cell division. CHO1 is thought to exert this effect by blocking the activity of a 110-kD KLP. The relevant target of HD, which was raised against the KHC motor domain, is unknown; HD may disrupt mitosis by interfering with an essential spindle KLP but not with KHC itself, as preabsorption of HD with KHC did not alter its ability to block mitosis. These data indicate that some KLPs have essential mitotic functions in early sea urchin embryos but KHC itself does not.

The kinesin-like protein KLP61F is essential for mitosis in Drosophila.

We report here that disruption of a recently discovered kinesin-like protein in Drosophila melanogaster, KLP61F, results in a mitotic mutation lethal to the organism. We show that in the absence of KLP61F function, spindle poles fail to separate, resulting in the formation of monopolar mitotic spindles. The resulting phenotype of metaphase arrest with polyploid cells is reminiscent of that seen in the fungal bimC and cut7 mutations, where it has also been shown that spindle pole bodies are not segregated. KLP61F is specifically expressed in proliferating tissues during embryonic and larval development, consistent with a primary role in cell division. The structural and functional homology of the KLP61F, bimC, cut7, and Eg5 kinesin-like proteins demonstrates the existence of a conserved family of kinesin-like molecules important for spindle pole separation and mitotic spindle dynamics.