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Sample records for kinesin spindle protein

  1. Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles

    PubMed Central

    1992-01-01

    We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis. PMID:1447302

  2. Kinesin spindle protein SiRNA slows tumor progression.

    PubMed

    Marra, Emanuele; Palombo, Fabio; Ciliberto, Gennaro; Aurisicchio, Luigi

    2013-01-01

    The kinesin spindle protein (KSP), a member of the kinesin superfamily of microtubule-based motors, plays a critical role in mitosis as it mediates centrosome separation and bipolar spindle assembly and maintenance. Inhibition of KSP function leads to cell cycle arrest at mitosis with the formation of monoastral microtubule arrays, and ultimately, to cell death. Several KSP inhibitors are currently being studied in clinical trials and provide new opportunities for the development of novel anticancer therapeutics. RNA interference (RNAi) may represent a powerful strategy to interfere with key molecular pathways involved in cancer. In this study, we have established an efficient method for intratumoral delivery of siRNA. We evaluated short interfering RNA (siRNA) duplexes targeting luciferase as surrogate marker or KSP sequence. To examine the potential feasibility of RNAi therapy, the siRNA was transfected into pre-established lesions by means of intratumor electro-transfer of RNA therapeutics (IERT). This technology allowed cell permeation of the nucleic acids and to efficiently knock down gene expression, albeit transiently. The KSP-specific siRNA drastically reduced outgrowth of subcutaneous melanoma and ovarian cancer lesions. Our results show that intratumoral electro-transfer of siRNA is feasible and KSP-specific siRNA may provide a novel strategy for therapeutic intervention.

  3. Acrylamide effects on kinesin-related proteins of the mitotic/meiotic spindle

    SciTech Connect

    Sickles, Dale W. . E-mail: dsickles@mcg.edu; Sperry, Ann O. . E-mail: sperrya@ecu.edu; Testino, Angie; Friedman, Marvin

    2007-07-01

    The microtubule (MT) motor protein kinesin is a vital component of cells and organs expressing acrylamide (ACR) toxicity. As a mechanism of its potential carcinogenicity, we determined whether kinesins involved in cell division are inhibited by ACR similar to neuronal kinesin [Sickles, D.W., Brady, S.T., Testino, A.R., Friedman, M.A., and Wrenn, R.A. (1996). Direct effect of the neurotoxicant acrylamide on kinesin-based microtubule motility. Journal of Neuroscience Research 46, 7-17.] Kinesin-related genes were isolated from rat testes [Navolanic, P.M., and Sperry, A.O. (2000). Identification of isoforms of a mitotic motor in mammalian spermatogenesis. Biology of Reproduction 62, 1360-1369.], their kinesin-like proteins expressed in bacteria using recombinant DNA techniques and the effects of ACR, glycidamide (GLY) and propionamide (a non-neurotoxic metabolite) on the function of two of the identified kinesin motors were tested. KIFC5A MT bundling activity, required for mitotic spindle formation, was measured in an MT-binding assay. Both ACR and GLY caused a similar concentration-dependent reduction in the binding of MT; concentrations of 100 {mu}M ACR or GLY reduced its activity by 60%. KRP2 MT disassembling activity was assayed using the quantity of tubulin disassembled from taxol-stabilized MT. Both ACR and GLY inhibited KRP2-induced MT disassembly. GLY was substantially more potent; significant reductions of 60% were achieved by 500 {mu}M, a comparable inhibition by ACR required a 5 mM concentration. Propionamide had no significant effect on either kinesin, except KRP2 at 10 mM. This is the first report of ACR inhibition of a mitotic/meiotic motor protein. ACR (or GLY) inhibition of kinesin may be an alternative mechanism to DNA adduction in the production of cell division defects and potential carcinogenicity. We conclude that ACR may act on multiple kinesin family members and produce toxicities in organs highly dependent on microtubule-based functions.

  4. Activity of the Kinesin Spindle Protein Inhibitor Ispinesib (SB-715992) in Models of Breast Cancer

    PubMed Central

    Purcell, James W.; Davis, Jefferson; Reddy, Mamatha; Martin, Shamra; Samayoa, Kimberly; Vo, Hung; Thomsen, Karen; Bean, Peter; Kuo, Wen Lin; Ziyad, Safiyyah; Billig, Jessica; Feiler, Heidi S.; Gray, Joe W.; Wood, Kenneth W.; Cases, Sylvaine

    2010-01-01

    Purpose Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein, a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have shown a 9% response rate in patients with locally advanced or metastatic breast cancer and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities, or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer, we explored the activity of ispinesib alone and in combination with several therapies approved for the treatment of breast cancer. Experimental Design We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo and tested the ability of ispinesib to enhance the antitumor activity of approved therapies. Results In vitro, ispinesib displayed broad antiproliferative activity against a panel of 53 breast cell lines. In vivo, ispinesib produced regressions in each of five breast cancer models and tumor-free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the antitumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine and exhibited activity comparable with paclitaxel and ixabepilone. Conclusions These findings support further clinical exploration of kinesin spindle protein inhibitors for the treatment of breast cancer. PMID:20068098

  5. Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer

    SciTech Connect

    Purcell, James W; Davis, Jefferson; Reddy, Mamatha; Martin, Shamra; Samayoa, Kimberly; Vo, Hung; Thomsen, Karen; Bean, Peter; Kuo, Wen Lin; Ziyad, Safiyyah; Billig, Jessica; Feiler, Heidi S; Gray, Joe W; Wood, Kenneth W; Cases, Sylvaine

    2009-06-10

    Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein (KSP), a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have demonstrated a 9% response rate in patients with locally advanced or metastatic breast cancer, and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer we explored the activity of ispinesib alone and in combination several therapies approved for the treatment of breast cancer. We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo, and tested the ability of ispinesib to enhance the anti-tumor activity of approved therapies. In vitro, ispinesib displayed broad anti-proliferative activity against a panel of 53 breast cell-lines. In vivo, ispinesib produced regressions in each of five breast cancer models, and tumor free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the anti-tumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine, and exhibited activity comparable to paclitaxel and ixabepilone. These findings support further clinical exploration of KSP inhibitors for the treatment of breast cancer.

  6. Kinesins to the core: the role of microtubule-based motor proteins in building the mitotic spindle midzone

    PubMed Central

    Hornick, Jessica E.; Karanjeet, Kul; Collins, Elizabeth S.; Hinchcliffe, Edward H.

    2013-01-01

    In mammalian cultured cells the initiation of cytokinesis is regulated – both temporally and spatially – by the overlapping, anti-parallel microtubules of the spindle midzone. This region recruits several key central spindle components: PRC-1, Polo-like kinase 1 (Plk-1), the centralspindlin complex, and the Chromosome Passenger Complex (CPC), which together serve to stabilize the microtubule overlap, and also to coordinate the assembly of the cortical actin/myosin cytoskeleton necessary to physically cleave the cell in two. The localization of these crucial elements to the spindle midzone requires members of the kinesin superfamily of microtubule-based motor proteins. Here we focus on reviewing the role played by a variety of kinesins in both building and operating the spindle midzone machinery during cytokinesis. PMID:20109573

  7. The Maize Divergent spindle-1 (dv1) Gene Encodes a Kinesin-14A Motor Protein Required for Meiotic Spindle Pole Organization

    PubMed Central

    Higgins, David M.; Nannas, Natalie J.; Dawe, R. Kelly

    2016-01-01

    The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation. PMID:27610117

  8. The Drosophila kinesin-like protein KLP67A is essential for mitotic and male meiotic spindle assembly.

    PubMed

    Gandhi, Rita; Bonaccorsi, Silvia; Wentworth, Diana; Doxsey, Stephen; Gatti, Maurizio; Pereira, Andrea

    2004-01-01

    We have performed a mutational analysis together with RNA interference to determine the role of the kinesin-like protein KLP67A in Drosophila cell division. During both mitosis and male meiosis, Klp67A mutations cause an increase in MT length and disrupt discrete aspects of spindle assembly, as well as cytokinesis. Mutant cells exhibit greatly enlarged metaphase spindle as a result of excessive MT polymerization. The analysis of both living and fixed cells also shows perturbations in centrosome separation, chromosome segregation, and central spindle assembly. These data demonstrate that the MT plus end-directed motor KLP67A is essential for spindle assembly during mitosis and male meiosis and suggest that the regulation of MT plus-end polymerization is a key determinant of spindle architecture throughout cell division.

  9. Distinct Kinesin-14 mitotic mechanisms in spindle bipolarity.

    PubMed

    Simeonov, Dimitre R; Kenny, Katelyn; Seo, Lan; Moyer, Amanda; Allen, Jessica; Paluh, Janet L

    2009-11-01

    Kinesin-like proteins are integral to formation and function of a conserved mitotic spindle apparatus that directs chromosome segregation and precedes cell division. Ubiquitous to the mechanism of spindle assembly and stability are balanced Kinesin-5 promoting and Kinesin-14 opposing forces. Distinct Kinesin-14 roles in bipolarity in eukaryotes have not been shown, but are suggested by gamma-tubulin-based pole interactions that affect establishment and by microtubule cross-linking and sliding that maintain bipolarity and spindle length. Distinct roles also imply specialized functional domains. By cross-species analysis of compatible mechanisms in establishing mitotic bipolarity we demonstrate that Kinesin-14 human HSET (HsHSET) functionally replaces Schizosaccharomyces pombe Pkl1 and its action is similarly blocked by mutation in a Kinesin-14 binding site on gamma-tubulin. Drosophila DmNcd localizes preferentially to bundled interpolar microtubules in fission yeast and does not replace SpPkl1. Analysis of twenty-six Kinesin-14 derivatives, including Tail, Stalk or Neck-Motor chimeras, for spindle localization, spindle assembly and mitotic progression defined critical domains. The Tail of SpPkl1 contains functional elements enabling its role in spindle assembly that are distinct from but transferable to DmNcd, whereas HsHSET function utilizes both Tail and Stalk features. Our analysis is the first to demonstrate distinct mechanisms between SpPkl1 and DmNcd, and reveal that HsHSET shares functional overlap in spindle pole mechanisms.

  10. Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle protein motor domain.

    PubMed

    Sheth, Payal R; Shipps, Gerald W; Seghezzi, Wolfgang; Smith, Catherine K; Chuang, Cheng-Chi; Sanden, David; Basso, Andrea D; Vilenchik, Lev; Gray, Kimberly; Annis, D Allen; Nickbarg, Elliott; Ma, Yao; Lahue, Brian; Herbst, Ronald; Le, Hung V

    2010-09-28

    Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP ATPase activity. The uncompetitive inhibition with respect to ATP is also confirmed by the requirement of nucleotide for binding of the compounds. After preliminary affinity optimization, the benzimidazole series exhibited distinctive antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters. Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding Ispinesib-resistant tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination therapy.

  11. XCTK2: A Kinesin-related Protein That Promotes Mitotic Spindle Assembly in Xenopus laevis Egg Extracts

    PubMed Central

    Walczak, Claire E.; Verma, Suzie; Mitchison, Timothy J.

    1997-01-01

    We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central α-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor- arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles. PMID:9049251

  12. Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation

    PubMed Central

    Frazer, Corey; Joshi, Monika; Delorme, Caroline; Davis, Darlene; Bennett, Richard J.

    2015-01-01

    Candida albicans is a major fungal pathogen whose virulence is associated with its ability to transition from a budding yeast form to invasive hyphal filaments. The kinesin-14 family member CaKar3 is required for transition between these morphological states, as well as for mitotic progression and karyogamy. While kinesin-14 proteins are ubiquitous, CaKar3 homologs in hemiascomycete fungi are unique because they form heterodimers with noncatalytic kinesin-like proteins. Thus, CaKar3-based motors may represent a novel antifungal drug target. We have identified and examined the roles of a kinesin-like regulator of CaKar3. We show that orf19.306 (dubbed CaCIK1) encodes a protein that forms a heterodimer with CaKar3, localizes CaKar3 to spindle pole bodies, and can bind microtubules and influence CaKar3 mechanochemistry despite lacking an ATPase activity of its own. Similar to CaKar3 depletion, loss of CaCik1 results in cell cycle arrest, filamentation defects, and an inability to undergo karyogamy. Furthermore, an examination of the spindle structure in cells lacking either of these proteins shows that a large proportion have a monopolar spindle or two dissociated half-spindles, a phenotype unique to the C. albicans kinesin-14 homolog. These findings provide new insights into mitotic spindle structure and kinesin motor function in C. albicans and identify a potentially vulnerable target for antifungal drug development. PMID:26024903

  13. Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle.

    PubMed Central

    Tokai, N; Fujimoto-Nishiyama, A; Toyoshima, Y; Yonemura, S; Tsukita, S; Inoue, J; Yamamota, T

    1996-01-01

    Microtubule-associated motor proteins are thought to be involved in spindle formation and chromosome movements in mitosis/meiosis. We have molecularly cloned cDNAs for a gene that codes for a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that the predicted gene product is a 73 kDa protein and is related to some extent to the Drosophila node gene product, which is involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, and its ability to bind to microtubules is demonstrated. Furthermore we show that its C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Accordingly, the protein was termed Kid (kinesin-like DNA binding protein). Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. Thus, we propose that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis. Images PMID:8599929

  14. A Kinesin-Related Protein, Krp180, Positions Prometaphase Spindle Poles during Early Sea Urchin Embryonic Cell Division

    PubMed Central

    Rogers, Gregory C.; Chui, Kitty K.; Lee, Edwin W.; Wedaman, Karen P.; Sharp, David J.; Holland, Gina; Morris, Robert L.; Scholey, Jonathan M.

    2000-01-01

    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. PMID:10931863

  15. Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein

    PubMed Central

    1992-01-01

    Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force. PMID:1740471

  16. Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes.

    PubMed

    Radford, Sarah J; Go, Allysa Marie M; McKim, Kim S

    2017-02-01

    The oocyte spindle in most animal species is assembled in the absence of the microtubule-organizing centers called centrosomes. Without the organization provided by centrosomes, acentrosomal meiotic spindle organization may rely heavily on the bundling of microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-14 NCD, and the plus-end directed kinesin-6 Subito are known to be required for oocyte spindle organization in Drosophila melanogaster How multiple microtubule-bundling kinesins interact to produce a functional acentrosomal spindle is not known. In addition, there have been few studies on the meiotic function of one of the most important microtubule-bundlers in mitotic cells, the kinesin-5 KLP61F. We have found that the kinesin-5 KLP61F is required for spindle and centromere symmetry in oocytes. The asymmetry observed in the absence of KLP61F depends on NCD, the kinesin-12 KLP54D, and the microcephaly protein ASP. In contrast, KLP61F and Subito work together in maintaining a bipolar spindle. We propose that the prominent central spindle, stabilized by Subito, provides the framework for the coordination of multiple microtubule-bundling activities. The activities of several proteins, including NCD, KLP54D, and ASP, generate asymmetries within the acentrosomal spindle, while KLP61F and Subito balance these forces, resulting in the capacity to accurately segregate chromosomes.

  17. An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis.

    PubMed

    Tao, Weikang; South, Victoria J; Diehl, Ronald E; Davide, Joseph P; Sepp-Lorenzino, Laura; Fraley, Mark E; Arrington, Kenneth L; Lobell, Robert B

    2007-01-01

    The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.

  18. An Inhibitor of the Kinesin Spindle Protein Activates the Intrinsic Apoptotic Pathway Independently of p53 and De Novo Protein Synthesis▿ †

    PubMed Central

    Tao, Weikang; South, Victoria J.; Diehl, Ronald E.; Davide, Joseph P.; Sepp-Lorenzino, Laura; Fraley, Mark E.; Arrington, Kenneth L.; Lobell, Robert B.

    2007-01-01

    The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors. PMID:17101792

  19. Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

    SciTech Connect

    Ying, Bo; Campbell, Robert B.

    2014-04-04

    Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carrier systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest amount of

  20. Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

    PubMed Central

    Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

    2016-01-01

    Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

  1. Motor proteins 1: kinesins.

    PubMed

    Bloom, G S; Endow, S A

    1995-01-01

    Progress regarding the kinesins is now being made at a rapid and accelerating rate. The in vivo-functions, and biophysical and enzymatic properties of kinesin itself are being explored at ever increasing levels of detail. The kinesin-related proteins now number several dozen, and although more is known about primary structure than function for most of the proteins, this trend is already reversing. For example, knowledge about the kinesin-related protein, ncd, is expanding rapidly, and more is already known about its three-dimensional structure than is known for kinesin heavy chain. This volume presents a comprehensive review of the major published works on kinesin and kinesin-related proteins. Hopefully, this manuscript will complement other recent review articles [17, 20, 25, 37, 60-62, 67, 69, 75, 85-88, 231, 233, 238, 244, 269-271, 281, 282, 292] or books [49, 227, 293] that have focused on more selective aspects of the kinesin family, or have been aimed more generally at MT motor proteins. In line with the stated purpose of the Protein Profile series, annual updates of the review on the kinesins are planned for at least the next few years.

  2. Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation.

    PubMed

    She, Zhen-Yu; Yang, Wan-Xi

    2017-07-01

    During eukaryote cell division, molecular motors are crucial regulators of microtubule organization, spindle assembly, chromosome segregation and intracellular transport. The kinesin-14 motors are evolutionarily conserved minus-end-directed kinesin motors that occur in diverse organisms from simple yeasts to higher eukaryotes. Members of the kinesin-14 motor family can bind to, crosslink or slide microtubules and, thus, regulate microtubule organization and spindle assembly. In this Commentary, we present the common subthemes that have emerged from studies of the molecular kinetics and mechanics of kinesin-14 motors, particularly with regard to their non-processive movement, their ability to crosslink microtubules and interact with the minus- and plus-ends of microtubules, and with microtubule-organizing center proteins. In particular, counteracting forces between minus-end-directed kinesin-14 and plus-end-directed kinesin-5 motors have recently been implicated in the regulation of microtubule nucleation. We also discuss recent progress in our current understanding of the multiple and fundamental functions that kinesin-14 motors family members have in important aspects of cell division, including the spindle pole, spindle organization and chromosome segregation. © 2017. Published by The Company of Biologists Ltd.

  3. Chromosome and mitotic spindle dynamics in fission yeast kinesin-8 mutants

    NASA Astrophysics Data System (ADS)

    Crapo, Ammon M.; Gergley, Zachary R.; McIntosh, J. Richard; Betterton, M. D.

    2014-03-01

    Fission yeast proteins Klp5p and Klp6p are plus-end directed motors of the kinesin-8 family which promote microtubule (MT) depolymerization and also affect chromosome segregation, but the mechanism of these activities is not well understood. Using live-cell time-lapse fluorescence microscopy of fission yeast wild-type (WT) and klp5/6 mutant strains, we quantify and compare the dynamics of kinetochore motion and mitotic spindle length in 3D. In WT cells, the spindle, once formed, remains a consistent size and chromosomes are correctly organized and segregated. In kinesin-8 mutants, spindles undergo large length fluctuations of several microns. Kinetochore motions are also highly fluctuating, with kinetochores frequently moving away from the spindle rather than toward it. We observe transient pushing of chromosomes away from the spindle by as much as 10 microns in distance.

  4. The novel kinesin spindle protein (KSP) inhibitor SB-743921 exhibits marked activity in in vivo and in vitro models of aggressive large B-cell lymphoma.

    PubMed

    Bongero, Danielle; Paoluzzi, Luca; Marchi, Enrica; Zullo, Kelly M; Neisa, Roberto; Mao, Yinghui; Escandon, Rafael; Wood, Ken; O'Connor, Owen A

    2015-01-01

    The kinesin spindle protein (KSP) is a mitotic protein essential for cell cycle control and motility. SB-743921 (hereafter SB-921) is an inhibitor that selectively targets the ATP-binding domain of the KSP. The preclinical activity of SB-921 was evaluated in models of diffuse large B-cell lymphoma (DLBCL). The cytotoxicity of SB-921 was evaluated in a series of germinal center (GC-DLBCL) and post-germinal center (ABC-DLBCL) DLBCL cell lines and a murine lymphoma xenograft model. GC-DLBCL lines generally demonstrated greater sensitivity to SB-921. IC50 values ranged between 1 nM and 900 nM for GC-DLBCL compared to 1 nM to 10 μM for ABC lines. SB-921 demonstrated marked activity in a xenograft model of Ly-1 (GC-DLBCL). While SB-921 was relatively more active in GC derived cell lines, ABC-derived lines still underwent apoptosis at higher concentrations. These results demonstrate that SB-921 inhibits proliferation and induces apoptosis in both GC-DLBCL and ABC-DLBCL.

  5. Thermodynamics of nucleotide and inhibitor binding to wild-type and ispinesib-resistant forms of human kinesin spindle protein.

    PubMed

    Sheth, Payal R; Basso, Andrea; Duca, José S; Lesburg, Charles A; Ogas, Polina; Gray, Kimberly; Nale, Lissette; Mannarino, Anthony F; Prongay, Andrew J; Le, Hung V

    2009-11-24

    Current antimitotic cancer chemotherapy based on vinca alkaloids and taxanes target tubulin, a protein required not only for mitotic spindle formation but also for the overall structural integrity of terminally differentiated cells. Among many innovations targeting specific mitotic events, inhibition of motor enzymes including KSP (or Eg5) has been validated as a highly productive approach. Many reported KSP inhibitors bind to an induced allosteric site near the site of ATP hydrolysis, and some have been tested in clinical trials with varying degrees of success. This allosteric site was defined in detail by X-ray crystallography of inhibitor complexes, yet complementary information on binding thermodynamics is still lacking. Using two model ATP-uncompetitive inhibitors, monastrol and ispinesib, we report here the results of thermal denaturation and isothermal titration calorimetric studies. These binding studies were conducted with the wild-type KSP motor domain as well as two ispinesib mutants (D130V and A133D) identified to confer resistance to ispinesib treatment. The thermodynamic parameters obtained were placed in the context of the available structural information and corresponding models of the two ispinesib-resistant mutants. The resulting overall information formed a strong basis for future structure-based design of inhibitors of KSP and related motor enzymes.

  6. The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

    PubMed Central

    Rizk, Rania S.; DiScipio, Katherine A.; Proudfoot, Kathleen G.

    2014-01-01

    Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation. PMID:24616221

  7. The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization.

    PubMed

    Rizk, Rania S; Discipio, Katherine A; Proudfoot, Kathleen G; Gupta, Mohan L

    2014-03-17

    Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.

  8. Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control.

    PubMed

    Su, Xiaolei; Arellano-Santoyo, Hugo; Portran, Didier; Gaillard, Jeremie; Vantard, Marylin; Thery, Manuel; Pellman, David

    2013-08-01

    Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide-disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

  9. Kinesin-1 Prevents Capture of the Oocyte Meiotic Spindle by the Sperm Aster

    PubMed Central

    McNally, Karen L.P.; Fabritius, Amy S.; Ellefson, Marina L.; Flynn, Jonathan R.; Milan, Jennifer A.; McNally, Francis J.

    2012-01-01

    Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a non-motor mechanism. PMID:22465668

  10. De novo design, synthesis and biological evaluation of 1,4-dihydroquinolin-4-ones and 1,2,3,4-tetrahydroquinazolin-4-ones as potent kinesin spindle protein (KSP) inhibitors.

    PubMed

    Jiang, Cheng; Yang, Lei; Wu, Wu-Tong; Guo, Qing-Long; You, Qi-Dong

    2011-09-15

    Kinesin spindle protein (KSP) inhibitors are a promising class of anticancer agents that cause mitotic arrest in cells from a failure to form functional bipolar mitotic spindles. Here, we report the design, synthesis and biological evaluation of a novel series of 1,4-dihydroquinolin-4-ones and 1,2,3,4-tetrahydroquinazolin-4-ones using de novo design method. The synthesized compound was evaluated and proved to have potent inhibitory activities in the KSP ATPase. Compounds 15j and 15p show potent inhibitory activities in cell proliferation assays. Preferred compound 15j markedly induced G2/M phase cell cycle arrest with characteristic monoastral spindles and subsequent cell death in A549 cells. In vivo evaluation of 15j on the growth of transplantable S180 sarcoma in mice suggested its therapeutic potential for further development.

  11. B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast

    PubMed Central

    Chee, Mark K.; Haase, Steven B.

    2010-01-01

    Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. PMID:20463882

  12. Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast

    NASA Astrophysics Data System (ADS)

    Rincon, Sergio A.; Lamson, Adam; Blackwell, Robert; Syrovatkina, Viktoriya; Fraisier, Vincent; Paoletti, Anne; Betterton, Meredith D.; Tran, Phong T.

    2017-05-01

    Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Δpkl1Δ spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Δpkl1Δ spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors.

  13. Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast

    PubMed Central

    Rincon, Sergio A.; Lamson, Adam; Blackwell, Robert; Syrovatkina, Viktoriya; Fraisier, Vincent; Paoletti, Anne; Betterton, Meredith D.; Tran, Phong T.

    2017-01-01

    Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Δpkl1Δ spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Δpkl1Δ spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors. PMID:28513584

  14. A novel small molecule inhibitor reveals a possible role of Kinesin-5 in anastral spindle pole assembly

    PubMed Central

    Groen, Aaron C; Needleman, Daniel; Brangwynne, Clifford; Gradinaru, Cristain; Fowler, Brandon; Mazitschek, Ralph; Mitchison, T. J.

    2010-01-01

    Summary The tetrameric plus-end directed motor, kinesin-5, is essential for bipolar spindle assembly. Small molecule inhibitors of kinesin-5 have been important tools for investigating its function, and some are currently under evaluation as anti-cancer drugs. Most inhibitors reported to date are “non-competitive inhibitors” and bind to a specific site on the motor head, trapping the motor in a state with ADP bound, and a weak but non-zero affinity for microtubules. Here we used a novel ATP competitive inhibitor, “FCPT”, developed at Merck (USA) which competes with the ATP substrate. We found that it induced tight binding of kinesin-5 onto microtubules in vitro. Using Xenopus egg extract spindles, we found FCPT not only blocks poleward microtubule sliding but also induced loss of microtubules selectively at the poles of bipolar spindles (and not asters or monoasters). We also found that the spindle pole proteins, TPX2 and γ-tubulin became redistributed to the spindle equator, suggesting proper kinesin-5 function is required for pole assembly. PMID:18559893

  15. Optimized S-trityl-L-cysteine-based inhibitors of kinesin spindle protein with potent in vivo antitumor activity in lung cancer xenograft models.

    PubMed

    Good, James A D; Wang, Fang; Rath, Oliver; Kaan, Hung Yi Kristal; Talapatra, Sandeep K; Podgórski, Dawid; MacKay, Simon P; Kozielski, Frank

    2013-03-14

    The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-L-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved potency, favorable drug-like properties, but moderate in vivo activity. We report here their further optimization to produce extremely potent inhibitors of Eg5 (K(i)(app) < 10 nM) with broad-spectrum activity against cancer cell lines comparable to the Phase II drug candidates ispinesib and SB-743921. They have good oral bioavailability and pharmacokinetics and induced complete tumor regression in nude mice explanted with lung cancer patient xenografts. Furthermore, they display fewer liabilities with CYP-metabolizing enzymes and hERG compared with ispinesib and SB-743921, which is important given the likely application of Eg5 inhibitors in combination therapies. We present the case for this preclinical series to be investigated in single and combination chemotherapies, especially targeting hematological malignancies.

  16. Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

    PubMed Central

    McCoy, Kelsey M.; Tubman, Emily S.; Claas, Allison; Tank, Damien; Clancy, Shelly Applen; O’Toole, Eileen T.; Berman, Judith; Odde, David J.

    2015-01-01

    A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to

  17. Kinesin-5 Contributes to Spindle-length Scaling in the Evolution of Cancer toward Metastasis

    PubMed Central

    Yang, Ching-Feng; Tsai, Wan-Yu; Chen, Wei-An; Liang, Kai-Wen; Pan, Cheng-Ju; Lai, Pei-Lun; Yang, Pan-Chyr; Huang, Hsiao-Chun

    2016-01-01

    During natural evolution, the spindles often scale with cell sizes to orchestrate accurate chromosome segregation. Whether in cancer evolution, when the constraints on genome integrity are relaxed, cancer cells may evolve the spindle to confer other advantages has not been investigated. Using invasion as a selective pressure in vitro, we found that a highly metastatic cancer clone displays a lengthened metaphase spindle, with faster spindle elongation that correlates with transiently elevated speed of cell migration. We found that kinesin-5 is upregulated in this malignant clone, and weak inhibition of kinesin-5 activity could revert the spindle to a smaller aspect ratio, decrease the speed of spindle pole separation, and suppress post-mitotic cell migration. A correlation was found between high aspect ratio and strong metastatic potential in cancers that evolved and were selected in vivo, implicating that the spindle aspect ratio could serve as a promising cellular biomarker for metastatic cancer clones. PMID:27767194

  18. Minus-end-directed Kinesin-14 motors align antiparallel microtubules to control metaphase spindle length.

    PubMed

    Hepperla, Austin J; Willey, Patrick T; Coombes, Courtney E; Schuster, Breanna M; Gerami-Nejad, Maryam; McClellan, Mark; Mukherjee, Soumya; Fox, Janet; Winey, Mark; Odde, David J; O'Toole, Eileen; Gardner, Melissa K

    2014-10-13

    During cell division, a microtubule-based mitotic spindle mediates the faithful segregation of duplicated chromosomes into daughter cells. Proper length control of the metaphase mitotic spindle is critical to this process and is thought to be achieved through a mechanism in which spindle pole separation forces from plus-end-directed motors are balanced by forces from minus-end-directed motors that pull spindle poles together. However, in contrast to this model, metaphase mitotic spindles with inactive kinesin-14 minus-end-directed motors often have shorter spindle lengths, along with poorly aligned spindle microtubules. A mechanistic explanation for this paradox is unknown. Using computational modeling, in vitro reconstitution, live-cell fluorescence microscopy, and electron microscopy, we now find that the budding yeast kinesin-14 molecular motor Kar3-Cik1 can efficiently align spindle microtubules along the spindle axis. This then allows plus-end-directed kinesin-5 motors to efficiently exert the outward microtubule sliding forces needed for proper spindle bipolarity. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed Central

    1993-01-01

    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. PMID:8227131

  20. A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes.

    PubMed

    Zou, Jianwei; Hallen, Mark A; Yankel, Christine D; Endow, Sharyn A

    2008-02-11

    The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.

  1. Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake

    PubMed Central

    Rozelle, Daniel K.; Hansen, Scott D.

    2011-01-01

    During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)–dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit. PMID:21482719

  2. Kinesin-5, a mitotic microtubule-associated motor protein, modulates neuronal migration

    PubMed Central

    Falnikar, Aditi; Tole, Shubha; Baas, Peter W.

    2011-01-01

    Kinesin-5 (also called Eg5 or kif11) is a homotetrameric motor protein that functions by modulating microtubule (MT)–MT interactions. In the case of mitosis, kinesin-5 slows the rate of separation of the half-spindles. In the case of the axon, kinesin-5 limits the frequency of transport of short MTs, and also limits the rate of axonal growth. Here we show that experimental inhibition of kinesin-5 in cultured migratory neurons results in a faster but more randomly moving neuron with a shorter leading process. As is the case with axons of stationary neurons, short MT transport frequency is notably enhanced in the leading process of the migratory neuron when kinesin-5 is inhibited. Conversely, overexpression of kinesin-5, both in culture and in developing cerebral cortex, causes migration to slow and even cease. Regions of anti-parallel MT organization behind the centrosome were shown to be especially rich in kinesin-5, implicating these regions as potential sites where kinesin-5 forces may be especially relevant. We posit that kinesin-5 acts as a “brake” on MT–MT interactions that modulates the advance of the entire MT apparatus. In so doing, kinesin-5 regulates the rate and directionality of neuronal migration and possibly the cessation of migration when the neuron reaches its destination. PMID:21411631

  3. Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity.

    PubMed

    Kevenaar, Josta T; Bianchi, Sarah; van Spronsen, Myrrhe; Olieric, Natacha; Lipka, Joanna; Frias, Cátia P; Mikhaylova, Marina; Harterink, Martin; Keijzer, Nanda; Wulf, Phebe S; Hilbert, Manuel; Kapitein, Lukas C; de Graaff, Esther; Ahkmanova, Anna; Steinmetz, Michel O; Hoogenraad, Casper C

    2016-04-04

    Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human neurological disorders. Homozygous nonsense mutations in kinesin-binding protein (KBP)/KIAA1279 cause the neurological disorder Goldberg-Shprintzen syndrome (GOSHS), which is characterized by intellectual disability, microcephaly, and axonal neuropathy. Here, we show that KBP regulates kinesin activity by interacting with the motor domains of a specific subset of kinesins to prevent their association with the MT cytoskeleton. The KBP-interacting kinesins include cargo-transporting motors such as kinesin-3/KIF1A and MT-depolymerizing motor kinesin-8/KIF18A. We found that KBP blocks KIF1A/UNC-104-mediated synaptic vesicle transport in cultured hippocampal neurons and in C. elegans PVD sensory neurons. In contrast, depletion of KBP results in the accumulation of KIF1A motors and synaptic vesicles in the axonal growth cone. We also show that KBP regulates neuronal MT dynamics by controlling KIF18A activity. Our data suggest that KBP functions as a kinesin inhibitor that modulates MT-based cargo motility and depolymerizing activity of a subset of kinesin motors. We propose that misregulation of KBP-controlled kinesin motors may represent the underlying molecular mechanism that contributes to the neuropathological defects observed in GOSHS patients.

  4. Minus-End Directed Kinesin-14 Motors Align Anti-Parallel Microtubules to Control Metaphase Spindle Length

    PubMed Central

    Hepperla, Austin J.; Willey, Patrick T.; Coombes, Courtney E.; Schuster, Breanna M.; Gerami-Nejad, Maryam; McClellan, Mark; Mukherjee, Soumya; Fox, Janet; Winey, Mark; Odde, David J.; O’Toole, Eileen; Gardner, Melissa K.

    2014-01-01

    Summary During cell division, a microtubule-based mitotic spindle mediates the faithful segregation of duplicated chromosomes into daughter cells. Proper length control of the metaphase mitotic spindle is critical to this process, and is thought to be achieved through a mechanism in which spindle pole separation forces from plus-end directed motors are balanced by forces from minus-end directed motors that pull spindle poles together. However, in contrast to this model, metaphase mitotic spindles with inactive Kinesin-14 minusend directed motors often have shorter spindle lengths, along with poorly aligned spindle microtubules. A mechanistic explanation for this paradox is unknown. Using computational modeling, in vitro reconstitution, live-cell fluorescence microscopy, and electron microscopy, we now find that the budding yeast Kinesin-14 molecular motor Kar3-Cik1 can efficiently align spindle microtubules along the spindle axis. This then allows plus-end directed Kinesin-5 motors to efficiently exert the outward microtubule sliding forces needed for proper spindle bipolarity. PMID:25313961

  5. Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes

    PubMed Central

    Das, Arunika; Shah, Shital J.; Fan, Bensen; Paik, Daniel; DiSanto, Daniel J.; Hinman, Anna Maria; Cesario, Jeffry M.; Battaglia, Rachel A.; Demos, Nicole; McKim, Kim S.

    2016-01-01

    Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule–chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation. PMID:26564158

  6. Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1

    PubMed Central

    Wolff, Ian D.; Tran, Michael V.; Mullen, Timothy J.; Villeneuve, Anne M.; Wignall, Sarah M.

    2016-01-01

    Although centrosomes contribute to spindle formation in most cell types, oocytes of many species are acentrosomal and must organize spindles in their absence. Here we investigate this process in Caenorhabditis elegans, detailing how acentrosomal spindles form and revealing mechanisms required to establish bipolarity. Using high-resolution imaging, we find that in meiosis I, microtubules initially form a “cage-like” structure inside the disassembling nuclear envelope. This structure reorganizes so that minus ends are sorted to the periphery of the array, forming multiple nascent poles that then coalesce until bipolarity is achieved. In meiosis II, microtubules nucleate in the vicinity of chromosomes but then undergo similar sorting and pole formation events. We further show that KLP-18/kinesin-12 and MESP-1, previously shown to be required for spindle bipolarity, likely contribute to bipolarity by sorting microtubules. After their depletion, minus ends are not sorted outward at the early stages of spindle assembly and instead converge. These proteins colocalize on microtubules, are interdependent for localization, and can interact, suggesting that they work together. We propose that KLP-18/kinesin-12 and MESP-1 form a complex that functions to sort microtubules of mixed polarity into a configuration in which minus ends are away from the chromosomes, enabling formation of nascent poles. PMID:27559133

  7. Engineered kinesin motor proteins amenable to small-molecule inhibition

    PubMed Central

    Engelke, Martin F.; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T. Lynne; Soppina, Pushpanjali; Hancock, William O.; Gelfand, Vladimir I.; Verhey, Kristen J.

    2016-01-01

    The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

  8. The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.

    PubMed

    Sharp, D J; McDonald, K L; Brown, H M; Matthies, H J; Walczak, C; Vale, R D; Mitchison, T J; Scholey, J M

    1999-01-11

    Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a "sliding filament mechanism" during the formation and function of the mitotic spindle.

  9. Phospho-regulated interaction between kinesin-6 Klp9p and microtubule bundler Ase1p promotes spindle elongation.

    PubMed

    Fu, Chuanhai; Ward, Jonathan J; Loiodice, Isabelle; Velve-Casquillas, Guilhem; Nedelec, Francois J; Tran, Phong T

    2009-08-01

    The spindle midzone-composed of antiparallel microtubules, microtubule-associated proteins (MAPs), and motors-is the structure responsible for microtubule organization and sliding during anaphase B. In general, MAPs and motors stabilize the midzone and motors produce sliding. We show that fission yeast kinesin-6 motor klp9p binds to the microtubule antiparallel bundler ase1p at the midzone at anaphase B onset. This interaction depends upon the phosphorylation states of klp9p and ase1p. The cyclin-dependent kinase cdc2p phosphorylates and its antagonist phosphatase clp1p dephosphorylates klp9p and ase1p to control the position and timing of klp9p-ase1p interaction. Failure of klp9p-ase1p binding leads to decreased spindle elongation velocity. The ase1p-mediated recruitment of klp9p to the midzone accelerates pole separation, as suggested by computer simulation. Our findings indicate that a phosphorylation switch controls the spatial-temporal interactions of motors and MAPs for proper anaphase B, and suggest a mechanism whereby a specific motor-MAP conformation enables efficient microtubule sliding.

  10. Phospho-regulated interaction between kinesin-6 klp9p and microtubule bundler ase1p promotes spindle elongation

    PubMed Central

    Fu, Chuanhai; Ward, Jonathan J.; Loiodice, Isabelle; Velve-Casquillas, Guilhem; Nedelec, Francois J.; Tran, Phong T.

    2010-01-01

    The spindle midzone – composed of antiparallel microtubules, microtubule-associated proteins (MAPs), and motors – is the structure responsible for microtubule organization and sliding during anaphase B. In general, MAPs and motors stabilize the midzone and motors produce sliding. We show that fission yeast kinesin-6 motor klp9p binds to the microtubule antiparallel bundler ase1p at the midzone at anaphase B onset. This interaction depends upon the phosphorylation states of klp9p and ase1p. The cyclin-dependent kinase cdc2p phosphorylates and its antagonist phosphatase clp1p dephosphorylates klp9p and ase1p to control the position and timing of klp9p-ase1p interaction. Failure of klp9p-ase1p binding leads to decreased spindle elongation velocity. The ase1p-mediated recruitment of klp9p to the midzone accelerates pole separation, as suggested by computer simulation. Our findings indicate that a phosphorylation switch controls the spatial-temporal interactions of motors and MAPs for proper anaphase B, and suggest a mechanism whereby a specific motor-MAP conformation enables efficient microtubule sliding. PMID:19686686

  11. Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle.

    PubMed

    Ito, Ami; Goshima, Gohta

    2015-12-07

    Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. We show that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. We identified a critical domain in Asp exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functioned as a global spindle coalescence factor not limited to MT ends. We propose a revised molecular model for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker.

  12. Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle

    PubMed Central

    Ito, Ami

    2015-01-01

    Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. We show that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. We identified a critical domain in Asp exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functioned as a global spindle coalescence factor not limited to MT ends. We propose a revised molecular model for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker. PMID:26644514

  13. Force generation by kinesin and myosin cytoskeletal motor proteins

    PubMed Central

    Kull, F. Jon; Endow, Sharyn A.

    2013-01-01

    Summary Kinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. How do motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles and conformational changes observed in crystal structures. Distortion or twisting of the central β-sheet – proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins – is shown in a movie depicting the transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governs ATP hydrolysis and Pi release by the motors, explaining the essential role of switch I in hydrolysis. Comparison of the motor power strokes reveals that each stroke begins with the force-amplifying structure oriented opposite to the direction of rotation or swing. Motors undergo changes in their mechanochemical cycles in response to small-molecule inhibitors, several of which bind to kinesins by induced fit, trapping the motors in a state that resembles a force-producing conformation. An unusual motor activator specifically increases mechanical output by cardiac myosin, potentially providing valuable information about its mechanism of function. Further study is essential to understand motor mechanochemical coupling and energy transduction, and could lead to new therapies to treat human disease. PMID:23487037

  14. Characterization of kinesin-like proteins in silkworm posterior silk gland cells.

    PubMed

    Wang, Qiao; Teng, Junlin; Shen, Birong; Zhang, Wei; Guo, Yige; Su, Xiaolei; Zhang, Chuanxi; Yu, Albert C H; Chen, Jianguo

    2010-06-01

    Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesins. Here, we report silkworm posterior silkgland (PSG), specialized cells with an extensive endomembrane system for intracellular transport and efficient secretion of fibroin, as a novel model for kinesin study. To investigate kinesin-driven intracellular transport in PSG cells, we cloned five silkworm kinesin-like proteins (KLPs), BmKinesin-1, BmKinesin-6, BmKinesin-7, BmKinesin-13, and BmKinesin-14A. We determined their expression patterns by relative real-time PCR and western blotting. Immunofluorescence microscopy verified their colocalization with microtubules. By combining pull-down assays, LC-MS/MS, and western blotting analysis, we identified many potential cargoes of BmKinesin-1 in PSG, including fibroin-containing granules and exuperantia-associated ribonucleoprotein (RNP) complexes. Moreover, BmKinesin-13 overexpression disrupted the microtubule network in BmN cells, which is consistent with a role of Kinesin-13 in regulating microtubule dynamics in other organisms. On the basis of these results, we concluded that PSG might have advantages in elucidating mechanisms of intracellular transport in secretory tissues and could serve as a potential model for kinesin studies.

  15. Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1

    PubMed Central

    Connolly, Amy A.; Osterberg, Valerie; Christensen, Sara; Price, Meredith; Lu, Chenggang; Chicas-Cruz, Kathy; Lockery, Shawn; Mains, Paul E.; Bowerman, Bruce

    2014-01-01

    In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions—ASPM-1 recruitment to the spindle and microtubule severing—both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity. PMID:24554763

  16. The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis

    SciTech Connect

    Castillo, Andrew; Justice, Monica J. . E-mail: mjustice@bcm.tmc.edu

    2007-06-08

    Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5-3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.

  17. An inner centromere protein that stimulates the microtubule depolymerizing activity of a KinI kinesin.

    PubMed

    Ohi, Ryoma; Coughlin, Margaret L; Lane, William S; Mitchison, Timothy J

    2003-08-01

    Mitosis requires precise control of microtubule dynamics. The KinI kinesin MCAK, a microtubule depolymerase, is critical for this regulation. In a screen to discover previously uncharacterized microtubule-associated proteins, we identified ICIS, a protein that stimulates MCAK activity in vitro. Consistent with this biochemical property, blocking ICIS function in Xenopus extracts with antibodies caused excessive microtubule growth and inhibited spindle formation. Prior to anaphase, ICIS localized in an MCAK-dependent manner to inner centromeres, the chromosomal region located in between sister kinetochores. From Xenopus extracts, ICIS coimmunoprecipitated MCAK and the inner centromere proteins INCENP and Aurora B, which are thought to promote chromosome biorientation. By immunoelectron microscopy, we found that ICIS is present on the surface of inner centromeres, placing it in an ideal location to depolymerize microtubules associated laterally with inner centromeres. At inner centromeres, MCAK-ICIS may destabilize these microtubules and provide a mechanism that prevents kinetochore-microtubule attachment errors.

  18. Identification and partial characterization of mitotic centromere- associated kinesin, a kinesin-related protein that associates with centromeres during mitosis

    PubMed Central

    1995-01-01

    Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message. PMID:7822426

  19. A Chromatin-associated Kinesin-related Protein Required for Normal Mitotic Chromosome Segregation in Drosophila

    PubMed Central

    Molina, Isabel; Baars, Sigrid; Brill, Julie A.; Hales, Karen G.; Fuller, Margaret T.; Ripoll, Pedro

    1997-01-01

    The tiovivo (tio) gene of Drosophila encodes a kinesin-related protein, KLP38B, that colocalizes with condensed chromatin during cell division. Wild-type function of the tio gene product KLP38B is required for normal chromosome segregation during mitosis. Mitotic cells in tio larval brains displayed circular mitotic figures, increased ploidy, and abnormal anaphase figures. KLP38B mRNA is maternally provided and expressed in cells about to undergo division. We propose that KLP38B, perhaps redundantly with other chromosome-associated microtubule motor proteins, contributes to interactions between chromosome arms and microtubules important for establishing bipolar attachment of chromosomes and assembly of stable bipolar spindles. PMID:9396743

  20. Phase Transitions of Spindle-Associated Protein Regulate Spindle Apparatus Assembly

    PubMed Central

    Jiang, Hao; Wang, Shusheng; Huang, Yuejia; He, Xiaonan; Cui, Honggang; Zhu, Xueliang; Zheng, Yixian

    2015-01-01

    Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks. PMID:26388440

  1. Mitotic Spindle Form and Function

    PubMed Central

    Winey, Mark; Bloom, Kerry

    2012-01-01

    The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine. PMID:22491889

  2. XCTK1: A Xenopus C-terminal Kinesin-like Protein

    NASA Technical Reports Server (NTRS)

    Winfree, Seth; Wilhelm, Heike; Sawyer, Alan; Karsenti, Eric; Mitchison, Tim; Walczak, Claire; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    XCTK1 is 97kDa kinesin-like protein homologous to FKIF2 and KIFC3. XCTK1 is present at picomolar levels in eggs, embryos and cultured cells in a soluble high-molecular weight complex that is not associated with membranes. XCKT1 localizes to centrosomes in Xenopus A6 cells. Anti-XCTK1 antibodies also localize to spindle poles when injected into A6 cells or when added to extracts during in vitro spindle assembly reactions. XCTK1 is associated with the center of taxol-induced microtubule asters in extracts. Therefore its localization to poles is dependent on microtubule minus-ends and not on centrosomes per se. Overexpression of XCTK1 leads to centrosome destruction in cultured cells. XCTK1 was tagged at either the N- or C-terminus and transfected into Xenopus A6 cells At low expression levels, XCTK1 associated with centrosomes. At higher levels, the protein localized to insoluble cytoplasmic structures. Gamma-tubulin staining was dramatically decreased from centrosomes or altogether absent. The centrosomal SPJ antigen colocalized with XCTK1-containing structures. Upon nocodozole treatment, microtubules failed to regrow from the centrosomes indicating that overexpression of XCTK1 severely compromises centrosomal function. Current studies are aimed at determining whether XCTK1 interacts directly with centrosomal proteins and to determine the effects of XCTK1 depletion on oocyte maturation and embryogenesis.

  3. XCTK1: A Xenopus C-terminal Kinesin-like Protein

    NASA Technical Reports Server (NTRS)

    Winfree, Seth; Wilhelm, Heike; Sawyer, Alan; Karsenti, Eric; Mitchison, Tim; Walczak, Claire; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    XCTK1 is 97kDa kinesin-like protein homologous to FKIF2 and KIFC3. XCTK1 is present at picomolar levels in eggs, embryos and cultured cells in a soluble high-molecular weight complex that is not associated with membranes. XCKT1 localizes to centrosomes in Xenopus A6 cells. Anti-XCTK1 antibodies also localize to spindle poles when injected into A6 cells or when added to extracts during in vitro spindle assembly reactions. XCTK1 is associated with the center of taxol-induced microtubule asters in extracts. Therefore its localization to poles is dependent on microtubule minus-ends and not on centrosomes per se. Overexpression of XCTK1 leads to centrosome destruction in cultured cells. XCTK1 was tagged at either the N- or C-terminus and transfected into Xenopus A6 cells At low expression levels, XCTK1 associated with centrosomes. At higher levels, the protein localized to insoluble cytoplasmic structures. Gamma-tubulin staining was dramatically decreased from centrosomes or altogether absent. The centrosomal SPJ antigen colocalized with XCTK1-containing structures. Upon nocodozole treatment, microtubules failed to regrow from the centrosomes indicating that overexpression of XCTK1 severely compromises centrosomal function. Current studies are aimed at determining whether XCTK1 interacts directly with centrosomal proteins and to determine the effects of XCTK1 depletion on oocyte maturation and embryogenesis.

  4. Phosphorylation-dependent protein interactions at the spindle midzone mediate cell cycle regulation of spindle elongation.

    PubMed

    Khmelinskii, Anton; Roostalu, Johanna; Roque, Helio; Antony, Claude; Schiebel, Elmar

    2009-08-01

    The metaphase-to-anaphase transition is one of the most dramatic and highly regulated steps in cell division. At anaphase onset the protease separase dissolves sister chromatid cohesion. Simultaneously, the mitotic spindle elongates as interpolar microtubules (iMTs) slide apart at the spindle midzone, ensuring chromosome segregation. However, it remains unclear how spindle elongation is coordinated with cell cycle progression. Here we demonstrate that phosphorylation of the midzone organizer Ase1 controls localization and function of Cin8, a kinesin-5 that slides iMTs relative to each other. Phosphorylation of Ase1 by Cdk1 (cyclin-dependent kinase) inhibits Cin8 binding to iMTs, preventing bending and collapse of the metaphase spindle. In anaphase Ase1 dephosphorylation by the separase-activated phosphatase Cdc14 is necessary and sufficient for Cin8 recruitment to the midzone, where it drives spindle elongation. Our results reveal that sliding forces at the midzone are activated by separase and explain how spindle elongation is triggered with anaphase entry.

  5. S-100 protein expressing spindle cells in spindle cell lipoma: a diagnostic pitfall.

    PubMed

    Mentzel, T; Rütten, A; Hantschke, M; Hornick, J L; Brenn, T

    2016-10-01

    Spindle cell lipoma represents a distinct clinicopathological entity and is related to cellular angiofibroma and mammary-type myofibroblastoma. Spindle cell lipomas are composed of mature lipogenic cells and a variable number of CD34-positive spindle cells that show loss of retinoblastoma protein expression. Spindle cell lipomas occasionally express S-100 protein. We studied one case of purely dermal spindle cell lipoma and four cases of classical subcutaneous spindle cell lipoma arising in one female and four male patients (age ranged from 55 to 69 years). The neoplasms arose on the nose, the chin, the neck, the forehead and retroauricular, and all lesions had been marginally or incompletely excised. The studied cases showed classical histological and immunohistochemical features of spindle cell lipoma and, in addition, strong expression of S-100 protein by spindle-shaped tumour cells. S-100-expression in spindle cell lipoma may cause problems in the differential diagnosis with neural and melanocytic neoplasms and emphasizes the plasticity of the spindle cells in spindle cell lipoma.

  6. Heterotrimeric Kinesin II Is the Microtubule Motor Protein Responsible for Pigment Dispersion in Xenopus Melanophores

    PubMed Central

    Tuma, M. Carolina; Zill, Andrew; Le Bot, Nathalie; Vernos, Isabelle; Gelfand, Vladimir

    1998-01-01

    Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720–3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles. PMID:9852150

  7. The novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein

    PubMed Central

    Wozniak, Marcin J; Melzer, Martina; Dorner, Cornelia; Haring, Hans-Ulrich; Lammers, Reiner

    2005-01-01

    Background Members of the Kinesin-3 family of kinesin-like proteins mediate transport of axonal vesicles (KIF1A, KIF1Bβ), distribution of mitochondria (KIF1Bα) and anterograde Golgi to ER vesicle transport (KIF1C). Until now, little is known about the regulation of kinesin-like proteins. Several proteins interact with members of this protein family. Here we report on a novel, KIF1 binding protein (KBP) that was identified in yeast two-hybrid screens. Results KBP was identified by using the yeast-two-hybrid system with an amino-terminal fragment of KIF1C as a bait that is strongly homologous to KIF1B. Here we investigated the interaction of KBP and KIF1B. The full length proteins coimmunoprecipitated after overexpression and in untransfected 293 cells. Immunofluorescence experiments revealed that KBP was mainly localized to mitochondria, as has been described for KIF1Bα. Overexpression of a deletion mutant or reduction of the KBP protein level using an anti-sense construct led to an aggregation of mitochondria. Such an effect is probably due to the lower activity of KIF1Bα in the absence of KBP, as was revealed in motility assays. Conclusion KBP is a new binding partner for KIF1Bα that is a regulator of its transport function and thus represents a new type of kinesin interacting protein. PMID:16225668

  8. Mobility of the von Hippel-Lindau tumour suppressor protein is regulated by kinesin-2.

    PubMed

    Mans, Dorus A; Lolkema, Martijn P; van Beest, Moniek; Daenen, Laura G; Voest, Emile E; Giles, Rachel H

    2008-04-01

    The von Hippel-Lindau tumour suppressor protein (pVHL) participates in many cellular processes including oxygen sensing, microtubule stability and primary cilia regulation. Recently, we identified ATP-dependent motor complex kinesin-2 to endogenously bind the full-length variant of VHL (pVHL30) in primary kidney cells, and mediate its association to microtubules. Here we show that pVHL also endogenously binds the neuronal kinesin-2 complex, which slightly differs from renal kinesin-2. To investigate the role of kinesin-2 in pVHL mobility, we performed fluorescence recovery after photobleaching (FRAP) experiments in neuroblastoma cells. We observe that pVHL30 is a highly mobile cytoplasmic protein, which becomes an immobile centrosomal protein after ATP-depletion in living cells. This response to ATP-depletion is independent of GSK3beta-dependent phosphorylation of pVHL30. Furthermore, VHL variant alleles with reduced binding to kinesin-2 fail to respond to ATP-depletion. Accordingly, interfering with pVHL30-KIF3A interaction by either overexpressing a dominant negative construct or by reducing endogenous cellular levels of KIF3A by RNAi abolishes pVHL's response to ATP-depletion. From these data we suggest that mobility of a subcellular pool of pVHL is regulated by the ATP-dependent kinesin-2 motor. Kinesin-2 driven mobility of cytoplasmic pVHL might enable pVHL to function as a tumour suppressor.

  9. Microtubule-binding protein doublecortin-like kinase 1 (DCLK1) guides kinesin-3-mediated cargo transport to dendrites.

    PubMed

    Lipka, Joanna; Kapitein, Lukas C; Jaworski, Jacek; Hoogenraad, Casper C

    2016-02-01

    In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule-binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin-3 (KIF1) and kinesin-4 (KIF21) subfamily that can also target dendrites. We found that microtubule-binding protein doublecortin-like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1-dependent dense-core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule-binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport. © 2016 The Authors.

  10. Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motor

    PubMed Central

    Tao, Li; Fasulo, Barbara; Warecki, Brandt; Sullivan, William

    2016-01-01

    Centralspindlin is essential for central spindle and cleavage furrow formation. Drosophila centralspindlin consists of a kinesin-6 motor (Pav/kinesin-6) and a GTPase-activating protein (Tum/RacGAP). Centralspindlin localization to the central spindle is mediated by Pav/kinesin-6. While Tum/RacGAP has well-documented scaffolding functions, whether it influences Pav/kinesin-6 function is less well-explored. Here we demonstrate that both Pav/kinesin-6 and the centralspindlin complex (co-expressed Pav/Tum) have strong microtubule bundling activity. Centralspindlin also has robust plus-end-directed motility. In contrast, Pav/kinesin-6 alone cannot move microtubules. However, the addition of Tum/RacGAP or a 65 amino acid Tum/RacGAP fragment to Pav/kinesin-6 restores microtubule motility. Further, ATPase assays reveal that microtubule-stimulated ATPase activity of centralspindlin is seven times higher than that of Pav/kinesin-6. These findings are supported by in vivo studies demonstrating that in Tum/RacGAP-depleted S2 Drosophila cells, Pav/kinesin-6 exhibits severely reduced localization to the central spindle and an abnormal concentration at the centrosomes. PMID:27091402

  11. The Cotton Kinesin-Like Calmodulin-Binding Protein Associates with Cortical Microtubles in Cotton Fibers

    SciTech Connect

    Preuss, Mary L.; Delmar, Deborah P.; Liu, Bo

    2003-05-01

    Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP was detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus the results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.

  12. TPX2 regulates neuronal morphology through kinesin-5 interaction

    PubMed Central

    Kahn, Olga I.; Ha, Ngoc; Baird, Michelle A.; Davidson, Michael W.; Baas, Peter W.

    2015-01-01

    TPX2 (targeting protein for Xklp2) is a multifunctional mitotic spindle assembly factor that in mammalian cells localizes and regulates mitotic motor protein kinesin-5 (also called Eg5 or kif11). We previously showed that upon depletion or inhibition of kinesin-5 in cultured neurons, microtubule movements increase, resulting in faster growing axons and thinner dendrites. Here, we show that depletion of TPX2 from cultured neurons speeds their rate of process outgrowth, similarly to kinesin-5 inhibition. The phenotype is rescued by TPX2 re-expression, but not if TPX2’s kinesin-5-interacting domain is deleted. These results, together with studies showing a spike in TPX2 expression during dendritic differentiation, suggest that the levels and distribution of TPX2 are likely to be determinants of when and where kinesin-5 acts in neurons. PMID:26257190

  13. The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system.

    PubMed

    Gindhart, Joseph G; Chen, Jinyun; Faulkner, Melissa; Gandhi, Rita; Doerner, Karl; Wisniewski, Tiffany; Nandlestadt, Aline

    2003-08-01

    Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.

  14. A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1pV⃞

    PubMed Central

    Paluh, Janet L.; Nogales, Eva; Oakley, Berl R.; McDonald, Kent; Pidoux, Alison L.; Cande, W. Z.

    2000-01-01

    Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component. PMID:10749926

  15. The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A.

    PubMed

    Brown, K D; Wood, K W; Cleveland, D W

    1996-05-01

    The kinesin-like protein CENP-E transiently associates with kinetochores following nuclear envelope breakdown in late prophase, remains bound throughout metaphase, but sometime after anaphase onset it releases and by telophase becomes bound to interzonal microtubules of the mitotic spindle. Inhibition of poleward chromosome movement in vitro by CENP-E antibodies and association of CENP-E with minus-end directed microtubule motility in vitro have combined to suggest a key role for CENP-E as an anaphase chromosome motor. For this to be plausible in vivo depends on whether CENP-E remains kinetochore associated during anaphase. Using Indian muntjac cells whose seven chromosomes have large, easily tracked kinetochores, we now show that CENP-E is kinetochore-associated throughout the entirety of anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to the interzonal microtubules. These observations support roles for CENP-E not only in the initial alignment of chromosomes at metaphase and in spindle elongation in anaphase-B, but also in poleward chromosome movement in anaphase-A.

  16. Functions of kinesin superfamily proteins in neuroreceptor trafficking.

    PubMed

    Wang, Na; Xu, Junyu

    2015-01-01

    Synaptic plasticity is widely regarded as the cellular basis of learning and memory. Understanding the molecular mechanism of synaptic plasticity has been one of center pieces of neuroscience research for more than three decades. It has been well known that the trafficking of α-amino-3-hydroxy-5-methylisoxazoloe-4-propionic acid- (AMPA-) type, N-methyl-D-aspartate- (NMDA-) type glutamate receptors to and from synapses is a key molecular event underlying many forms of synaptic plasticity. Kainate receptors are another type of glutamate receptors playing important roles in synaptic transmission. In addition, GABA receptors also play important roles in modulating the synaptic plasticity. Kinesin superfamily proteins (also known as KIFs) transport various cargos in both anterograde and retrograde directions through the interaction with different adaptor proteins. Recent studies indicate that KIFs regulate the trafficking of NMDA receptors, AMPA receptors, kainate receptors, and GABA receptors and thus play important roles in neuronal activity. Here we review the essential functions of KIFs in the trafficking of neuroreceptor and synaptic plasticity.

  17. Human microcephaly ASPM protein is a spindle pole-focusing factor that functions redundantly with CDK5RAP2.

    PubMed

    Tungadi, Elsa A; Ito, Ami; Kiyomitsu, Tomomi; Goshima, Gohta

    2017-09-07

    Nonsense mutations in the ASPM gene have been most frequently identified among familial microcephaly patients. Depletion of the Drosophila orthologue causes spindle pole unfocusing during mitosis in multiple cell types. However, it remains unknown whether human ASPM has a similar function. Here, using CRISPR-based gene knockout (KO) and RNA interference combined with auxin-inducible degron, we show that ASPM functions in spindle pole organisation during mitotic metaphase redundantly with another microcephaly protein CDK5RAP2 (also called CEP215) in human tissue culture cells. Deletion of the ASPM gene alone did not affect spindle morphology or mitotic progression. However, when the pericentriolar material protein CDK5RAP2 was depleted in ASPM KO cells, spindle poles were unfocused during prometaphase and anaphase onset was significantly delayed. The phenotypic analysis of CDK5RAP2-depleted cells suggested that the pole-focusing function of CDK5RAP2 is independent of its known function to localise the kinesin-14 motor HSET or activate the γ-tubulin complex. Finally, a hypomorphic mutation identified in ASPM microcephaly patients similarly caused spindle pole unfocusing in the absence of CDK5RAP2, suggesting a possible link between spindle pole disorganisation and microcephaly. © 2017. Published by The Company of Biologists Ltd.

  18. The spindle protein CHICA mediates localization of the chromokinesin Kid to the mitotic spindle.

    PubMed

    Santamaria, Anna; Nagel, Susanna; Sillje, Herman H W; Nigg, Erich A

    2008-05-20

    Microtubule-based motor proteins provide essential forces for bipolar organization of spindle microtubules and chromosome movement, prerequisites of chromosome segregation during the cell cycle. Here, we describe the functional characterization of a novel spindle protein, termed "CHICA," that was originally identified in a proteomic survey of the human spindle apparatus [1]. We show that CHICA localizes to the mitotic spindle and is both upregulated and phosphorylated during mitosis. CHICA-depleted cells form shorter spindles and fail to organize a proper metaphase plate, highly reminiscent of the phenotype observed upon depletion of the chromokinesin Kid, a key mediator of polar ejection forces [2-6]. We further show that CHICA coimmunoprecipitates with Kid and is required for the spindle localization of Kid without affecting its chromosome association. Moreover, upon depletion of either CHICA or Kid (or both proteins simultaneously), chromosomes collapse onto the poles of monastrol-induced monopolar spindles. We conclude that CHICA represents a novel interaction partner of the chromokinesin Kid that is required for the generation of polar ejection forces and chromosome congression.

  19. Dynamic Positioning of Mitotic Spindles in Yeast:

    PubMed Central

    Yeh, Elaine; Yang, Charlie; Chin, Elaine; Maddox, Paul; Salmon, E. D.; Lew, Daniel J.; Bloom, Kerry

    2000-01-01

    In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being “pushed” by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus “pulling” the nucleus toward the bud neck. Failure of “pulling” is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud. PMID:11071919

  20. A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

    NASA Technical Reports Server (NTRS)

    Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

    1996-01-01

    Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

  1. Spindle

    SciTech Connect

    2013-04-04

    Spindle is software infrastructure that solves file system scalabiltiy problems associated with starting dynamically linked applications in HPC environments. When an HPC applications starts up thousands of pricesses at once, and those processes simultaneously access a shared file system to look for shared libraries, it can cause significant performance problems for both the application and other users. Spindle scalably coordinates the distribution of shared libraries to an application to avoid hammering the shared file system.

  2. F-actin asymmetry and the endoplasmic reticulum-associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo.

    PubMed

    Berends, Christian W H; Muñoz, Javier; Portegijs, Vincent; Schmidt, Ruben; Grigoriev, Ilya; Boxem, Mike; Akhmanova, Anna; Heck, Albert J R; van den Heuvel, Sander

    2013-07-01

    The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.

  3. Temperature dependent properties of a kinesin-3 motor protein from Thermomyces lanuginosus.

    PubMed

    Rivera, Susan B; Koch, Steven J; Bauer, Joseph M; Edwards, J Matthew; Bachand, George D

    2007-11-01

    Kinesins are cytoskeletal motor proteins that share a common mechanochemical motor domain, and are responsible for trafficking macromolecules. Here we report the cloning and characterization of a monomeric, kinesin-3 (TKIN) from Thermomyces lanuginosus. TKIN displayed a maximum rate of ATP hydrolysis at approximately 55 degrees C; the K(m)(ATP) was also significantly greater at 50 degrees C. Gliding motility rates reached a maximum of 5.5 microms(-1) at 45 degrees C, which is among the highest rates reported for kinesin. Arrhenius energy barriers were calculated to be approximately 103 kJmol(-1), nearly twofold greater than other mesophilic kinesin motors. The enthalpy of activation and entropy activation of TKIN were also significantly greater when compared to other mesophilic kinesins. A thermally induced aggregation of TKIN, which could be moderated by the addition of ATP, was observed at temperatures above 45 degrees C. Together, these results illustrate the kinetic response and stability of this unique motor protein at elevated temperatures.

  4. Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage.

    PubMed

    Tao, Weikang; South, Victoria J; Zhang, Yun; Davide, Joseph P; Farrell, Linda; Kohl, Nancy E; Sepp-Lorenzino, Laura; Lobell, Robert B

    2005-07-01

    The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.

  5. Cell cycle regulation of central spindle assembly.

    PubMed

    Mishima, Masanori; Pavicic, Visnja; Grüneberg, Ulrike; Nigg, Erich A; Glotzer, Michael

    2004-08-19

    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). Here we describe 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.

  6. Tau proteins harboring neurodegeneration-linked mutations impair kinesin translocation in vitro.

    PubMed

    Yu, Dezhi; LaPointe, Nichole E; Guzman, Elmer; Pessino, Veronica; Wilson, Leslie; Feinstein, Stuart C; Valentine, Megan T

    2014-01-01

    We tested the hypothesis that mutant tau proteins that cause neurodegeneration and dementia differentially alter kinesin translocation along microtubules (MTs) relative to normal tau in vitro. We employed complementary in vitro motility assays using purified recombinant kinesin, purified recombinant tau, and purified bovine brain α:β tubulin to isolate interactions among these components without any contribution by cellular regulatory mechanisms. We found that kinesin translocates slower along MTs assembled by any of three independent tau mutants (4-repeat P301L tau, 4-repeat ΔN296 tau, and 4-repeat R406W tau) relative to its translocation rate along MTs assembled by normal, 4-repeat wild type (WT) tau. Moreover, the R406W mutation exhibited isoform specific effects; while kinesin translocation along 4-repeat R406W tau assembled MTs is slower than along MTs assembled by 4-repeat WT tau, the R406W mutation had no effect in the 3-repeat tau context. These data provide strong support for the notion that aberrant modulation of kinesin translocation is a component of tau-mediated neuronal cell death and dementia. Finally, we showed that assembling MTs with taxol before coating them with mutant tau obscured effects of the mutant tau that were readily apparent using more physiologically relevant MTs assembled with tau alone, raising important issues regarding the use of taxol as an experimental reagent and novel insights into therapeutic mechanisms of taxol action.

  7. The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm.

    PubMed

    Henson, J H; Cole, D G; Roesener, C D; Capuano, S; Mendola, R J; Scholey, J M

    1997-01-01

    We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression and localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred to as KRP(85/95)), in sea urchin and sand dollar sperm. We demonstrate the presence of the 85 K and 115 K subunits of kinesin-II in sperm and localize these proteins to the sperm flagella and midpiece. The kinesin-II localization pattern is punctate and discontinuous, and in the flagella it is quite distinct from the continuous labeling present in sperm labeled with anti-flagellar dynein. The kinesin-II staining is largely insensitive to prefixation detergent extraction, suggesting that it is not associated with membranous elements in the sperm. In the midpiece the kinesin-II staining is similar to the pattern present in sperm labeled with an anti-centrosomal antibody. To our knowledge, this is the first localization of kinesin-like proteins in mature sperm and corroborates the recent identification and localization of kinesin-like proteins in the flagella and basal body of the unicellular green alga Chlamydomonas. We hypothesize that kinesin-II in the sperm may play functional roles in intraflagellar transport and/or the formation of flagella during spermatogenesis.

  8. Heat-shock protein 70 binds microtubules and interacts with kinesin in tobacco pollen tubes.

    PubMed

    Parrotta, Luigi; Cresti, Mauro; Cai, Giampiero

    2013-09-01

    The heat-shock proteins of 70 kDa are a family of ubiquitously expressed proteins important for protein folding. Heat-shock protein 70 assists other nascent proteins to achieve the spatial structure and ultimately helps the cell to protect against stress factors, such as heat. These proteins are localized in different cellular compartments and are associated with the cytoskeleton. We identified a heat-shock protein 70 isoform in the pollen tube of tobacco that binds to microtubules in an ATP-dependent manner. The heat-shock protein 70 was identified as part of the so-called ATP-MAP (ATP-dependent microtubule-associated protein) fraction, which also includes the 90-kDa kinesin, a mitochondria-associated motor protein. The identity of heat-shock protein 70 was validated by immunological assays and mass spectrometry. Sequence analysis showed that this heat-shock protein 70 is more similar to specific heat-shock proteins of Arabidopsis than to corresponding proteins of tobacco. Two-dimensional electrophoresis indicated that this heat-shock protein 70 isoform only is part of the ATP-MAP fraction and that is associated with the mitochondria of pollen tubes. Sedimentation assays showed that the binding of heat-shock protein 70 to microtubules is not affected by AMPPNP but it increases in the presence of the 90-kDa kinesin. Binding of heat-shock protein 70 to microtubules occurs only partially in the presence of ATP but it does not occur if, in addition to ATP, the 90-kDa kinesin is also present. Data suggest that the binding (but not the release) of heat-shock protein 70 to microtubules is facilitated by the 90-kDa kinesin. Copyright © 2013 Wiley Periodicals, Inc.

  9. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation

    PubMed Central

    1991-01-01

    A novel protein has been identified which may serve a key function in nucleating spindle microtubule growth in mitosis. This protein, called centrophilin, is sequentially relocated from the centromeres to the centrosomes to the midbody in a manner dependent on the mitotic phase. Centrophilin was initially detected by immunofluorescence with a monoclonal, primate-specific antibody (2D3) raised against kinetochore- enriched chromosome extract from HeLa cells (Valdivia, M. M., and B. R. Brinkley. 1985. J. Cell Biol. 101:1124-1134). Centrophilin forms prominent crescents at the poles of the metaphase spindle, gradually diminishes during anaphase, and bands the equatorial ends of midbody microtubules in telophase. The formation and breakdown of the spindle and midbody correlates in time and space with the aggregation and disaggregation of centrophilin foci. Immunogold EM reveals that centrophilin is a major component of pericentriolar material in metaphase. During recovery from microtubule inhibition, centrophilin foci act as nucleation sites for the assembly of spindle tubules. The 2D3 probe recognizes two high molecular mass polypeptides, 180 and 210 kD, on immunoblots of whole HeLa cell extract. Taken together, these data and the available literature on microtubule dynamics point inevitably to a singular model for control of spindle tubule turnover. PMID:1991791

  10. Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts

    PubMed Central

    Kullmann , Frank; Judex , Martin; Ballhorn , Wibke; Jüsten , Hans-Peter; Wessinghage , Dieter; Welsh, John; Yen, Tim J; Lang , Bernhard; Hittle, Jim C; McClelland, Michael; Gay, Steffen; Schölmerich, Jürgen; Müller-Ladner, Ulf

    1999-01-01

    populations from RA patients receiving steroids, four showed CENP-E upregulation. Numerous synovial cells of the patients with RA showed a positive in situ signal for the isolated CENP-E gene segment, confirming CENP-E mRNA production in rheumatoid synovium, whereas in OA synovial tissue CENP-E mRNA could not be detected. In addition, CENP-E expression was independent from medication. This was further confirmed by analysis of the effect of prednisolone on CENP-E expression, which revealed no alteration in CENP-E mRNA after exposure to different (physiological) concentrations of prednisolone. Serum starvation also could not suppress CENP-E mRNA completely. Discussion: Since its introduction in 1992, numerous variants of the differential display method and continuous improvements including RAP-PCR have proved to have both efficiency and reliability in examination of differentially regulated genes. The results of the present study reveal that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. The mRNA, which has been found to be upregulated in rheumatoid synovial fibroblasts, codes for a kinesin-like motor protein named CENP-E, which was first characterized in 1991. It is a member of a family of centromere-associated proteins, of which six (CENP-A to CENP-F) are currently known. CENP-E itself is a kinetochore motor, which accumulates transiently at kinetochores in the G2 phase of the cell cycle before mitosis takes place, appears to modulate chromosome movement and spindle elongation,and is degraded at the end of mitosis. The presence or upregulation of CENP-E has never been associated with RA. The three-dimensional structure of CENP-E includes a coiled-coil domain. This has important functions and shows links to known pathways in RA pathophysiology. Coiled-coil domains can also be found in jun and fos oncogene products, which are frequently upregulated in RA synovial fibroblasts. They are also involved in DNA binding and

  11. Two kinesin-like proteins mediate actin-based chloroplast movement in Arabidopsis thaliana.

    PubMed

    Suetsugu, Noriyuki; Yamada, Noboru; Kagawa, Takatoshi; Yonekura, Hisashi; Uyeda, Taro Q P; Kadota, Akeo; Wada, Masamitsu

    2010-05-11

    Organelle movement is essential for efficient cellular function in eukaryotes. Chloroplast photorelocation movement is important for plant survival as well as for efficient photosynthesis. Chloroplast movement generally is actin dependent and mediated by blue light receptor phototropins. In Arabidopsis thaliana, phototropins mediate chloroplast movement by regulating short actin filaments on chloroplasts (cp-actin filaments), and the chloroplast outer envelope protein CHUP1 is necessary for cp-actin filament accumulation. However, other factors involved in cp-actin filament regulation during chloroplast movement remain to be determined. Here, we report that two kinesin-like proteins, KAC1 and KAC2, are essential for chloroplasts to move and anchor to the plasma membrane. A kac1 mutant showed severely impaired chloroplast accumulation and slow avoidance movement. A kac1kac2 double mutant completely lacked chloroplast photorelocation movement and showed detachment of chloroplasts from the plasma membrane. KAC motor domains are similar to those of the kinesin-14 subfamily (such as Ncd and Kar3) but do not have detectable microtubule-binding activity. The C-terminal domain of KAC1 could interact with F-actin in vitro. Instead of regulating microtubules, KAC proteins mediate chloroplast movement via cp-actin filaments. We conclude that plants have evolved a unique mechanism to regulate actin-based organelle movement using kinesin-like proteins.

  12. Axonal pruning is actively regulated by the microtubule-destabilizing protein kinesin superfamily protein 2A.

    PubMed

    Maor-Nof, Maya; Homma, Noriko; Raanan, Calanit; Nof, Aviv; Hirokawa, Nobutaka; Yaron, Avraham

    2013-04-25

    Extensive axonal pruning and neuronal cell death are critical events for the development of the nervous system. Like neuronal cell death, axonal elimination occurs in discrete steps; however, the regulators of these processes remain mostly elusive. Here, we identify the kinesin superfamily protein 2A (KIF2A) as a key executor of microtubule disassembly and axonal breakdown during axonal pruning. Knockdown of Kif2a, but not other microtubule depolymerization or severing proteins, protects axonal microtubules from disassembly upon trophic deprivation. We further confirmed and extended this result to demonstrate that the entire degeneration process is delayed in neurons from the Kif2a knockout mice. Finally, we show that the Kif2a-null mice exhibit normal sensory axon patterning early during development, but abnormal target hyperinnervation later on, as they compete for limited skin-derived trophic support. Overall, these findings reveal a central regulatory mechanism of axonal pruning during development.

  13. In vivo collection of rare proteins using kinesin-based "nano-harvesters".

    SciTech Connect

    Bachand, Marlene; Bachand, George David; Greene, Adrienne Celeste; Carroll-Portillo, Amanda

    2008-11-01

    In this project, we have developed a novel platform for capturing, transport, and separating target analytes using the work harnessed from biomolecular transport systems. Nanoharvesters were constructed by co-organizing kinesin motor proteins and antibodies on a nanocrystal quantum dot (nQD) scaffold. Attachment of kinesin and antibodies to the nQD was achieved through biotin-streptavidin non-covalent bonds. Assembly of the nanoharvesters was characterized using a modified enzyme-linked immunosorbent assay (ELISA) that confirmed attachment of both proteins. Nanoharvesters selective against tumor necrosis factor-{alpha} (TNF-{alpha}) and nuclear transcription factor-{kappa}B (NF-{kappa}B) were capable of detecting target antigens at <100 ng/mL in ELISAs. A motility-based assay was subsequently developed using an antibody-sandwich approach in which the target antigen (TNF-{alpha}) formed a sandwich with the red-emitting nanoharvester and green-emitting detection nQD. In this format, successful sandwich formation resulted in a yellow emission associated with surface-bound microtubules. Step-wise analysis of sandwich formation suggested that the motility function of the kinesin motors was not adversely affected by either antigen capture or the subsequent binding of the detection nQDs. TNF-{alpha} was detected as low as {approx}1.5 ng/mL TNF-{alpha}, with 5.2% of the nanoharvesters successfully capturing the target analyte and detection nQDs. Overall, these results demonstrate the ability to capture target protein analytes in vitro using the kinesin-based nanoharvesters in nanofluidic environments. This system has direct relevance for lab-on-a-chip applications where pressure-driven or electrokinetic movement of fluids is impractical, and offers potential application for in vivo capture of rare proteins within the cytoplasmic domain of live cells.

  14. Kinesin motors in plants: from subcellular dynamics to motility regulation.

    PubMed

    Lee, Yuh-Ru Julie; Qiu, Weihong; Liu, Bo

    2015-12-01

    Plants produce enormous forms of the microtubule (MT)-based motor kinesins that have been inspiring plant cell biologists to uncover their functions in relation to plant growth and development. Subcellular localization of kinesin proteins detected through live-cell imaging or immunofluorescence microscopy has provided great insights into the functions of these motors. Dozens of mitotic kinesins exhibit particularly splendid localization patterns from chromosomes and kinetochores to MT arrays like the preprophase band, spindle poles, the spindle midzone, phragmoplast distal ends, and the phragmoplast midzone. Different subcellular localizations indicate distinct functions of these motors that are yet to be characterized. The localization difference between plant kinesins and their animal counterparts implies mechanistic differences in mitosis and cytokinesis between the two kingdoms. When many forms of kinesins are present simultaneously, it becomes critical that their motility is differentially regulated with spatial and temporal precision. Insights into regulatory mechanisms of motors can often be brought about by in vitro single-molecule biophysical studies. Significant advances are expected in this area in the coming years owing to rapid technological advances that are being brought to various model plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65

    NASA Astrophysics Data System (ADS)

    Pringle, Joshua; Muthukumar, Amutha; Tan, Amanda; Crankshaw, Laura; Conway, Leslie; Ross, Jennifer L.

    2013-09-01

    Microtubules are rigid, proteinaceous filaments required to organize and rearrange the interior of cells. They organize space by two mechanisms, including acting as the tracks for long-distance cargo transporters, such as kinesin-1, and by forming a network that supports the shape of the cell. The microtubule network is composed of microtubules and a bevy of associated proteins and enzymes that self-organize using non-equilibrium dynamic processes. In order to address the effects of self-organization of microtubules, we have utilized the filament-gliding assay with kinesin-1 motors driving microtubule motion. To further enhance the complexity of the system and determine if new patterns are formed, we added the microtubule crosslinking protein MAP65-1. MAP65-1 is a microtubule-associated protein from plants that crosslinks antiparallel microtubules, similar to mammalian PRC1 and fission yeast Ase1. We find that MAP65 can slow and halt the velocity of microtubules in gliding assays, but when pre-formed microtubule bundles are added to gliding assays, kinesin-1 motors can pull apart the bundles and reconstitute cell-like protrusions.

  16. Polycystic kidney disease protein fibrocystin localizes to the mitotic spindle and regulates spindle bipolarity.

    PubMed

    Zhang, Jingjing; Wu, Maoqing; Wang, Shixuan; Shah, Jagesh V; Wilson, Patricia D; Zhou, Jing

    2010-09-01

    Autosomal recessive polycystic kidney disease (ARPKD) is a significant hereditary renal disease occurring in infancy and childhood, which presents with greatly enlarged echogenic kidneys, ultimately leading to renal insufficiency and end-stage renal disease. ARPKD is caused by mutations in a single gene PKHD1, which encodes fibrocystin/polyductin (FPC), a large single transmembrane protein generally known to be on the primary cilium, basal body and plasma membrane. Here, using our newly generated antibody raised against the entire C-terminal intracellular cytoplasmic domain (ICD) of FPC, as well as our previously well-characterized antibody against a peptide of ICD, we report for the first time that at least one isoform of FPC is localized to the centrosome and mitotic spindle of dividing cells in multiple cell lines, including MDCK, mIMCD3, LLC-PK1, HEK293, RCTEC and HFCT cells. Using short-hairpin-mediated RNA interference, we show that the inhibition of FPC function in MDCK and mIMCD3 cells leads to centrosome amplification, chromosome lagging and multipolar spindle formation. Consistent with our in vitro findings, we also observed centrosome amplification in the kidneys from human ARPKD patients. These findings demonstrate a novel function of FPC in centrosome duplication and mitotic spindle assembly during cell division. We propose that mitotic defects due to FPC dysfunction contribute to cystogenesis in ARPKD.

  17. Kinetochore localization of spindle checkpoint proteins: who controls whom?

    PubMed

    Vigneron, Suzanne; Prieto, Susana; Bernis, Cyril; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

    2004-10-01

    The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.

  18. Kinesin-5 inhibitor resistance is driven by kinesin-12

    PubMed Central

    Sturgill, Emma G.; Norris, Stephen R.; Guo, Yan

    2016-01-01

    The microtubule (MT) cytoskeleton bipolarizes at the onset of mitosis to form the spindle. In animal cells, the kinesin-5 Eg5 primarily drives this reorganization by actively sliding MTs apart. Its primacy during spindle assembly renders Eg5 essential for mitotic progression, demonstrated by the lethal effects of kinesin-5/Eg5 inhibitors (K5Is) administered in cell culture. However, cultured cells can acquire resistance to K5Is, indicative of alternative spindle assembly mechanisms and/or pharmacological failure. Through characterization of novel K5I-resistant cell lines, we unveil an Eg5 motility-independent spindle assembly pathway that involves both an Eg5 rigor mutant and the kinesin-12 Kif15. This pathway centers on spindle MT bundling instead of Kif15 overexpression, distinguishing it from those previously described. We further show that large populations (∼107 cells) of HeLa cells require Kif15 to survive K5I treatment. Overall, this study provides insight into the functional plasticity of mitotic kinesins during spindle assembly and has important implications for the development of antimitotic regimens that target this process. PMID:27091450

  19. Kinesin-14: the roots of reversal.

    PubMed

    Cross, Robert A

    2010-08-16

    Kinesin-14 motor proteins step towards microtubule minus ends, in the opposite direction to other kinesins. Work on the still-enigmatic kinesin-14 mechanism published in BMC Structural Biology shows that the carboxyl terminus of the motor head undergoes a dock-undock cycle, like that of plus-end-directed kinesins.

  20. Dynamic model for kinesin-mediated long-range transport and its local traffic jam caused by tau proteins

    NASA Astrophysics Data System (ADS)

    Nam, Woochul; Epureanu, Bogdan I.

    2017-01-01

    In neurons, several intracellular cargoes are transported by motor proteins (kinesins) which walk on microtubules (MTs). However, kinesins can possibly unbind from the MTs before they reach their destinations. The unbound kinesins randomly diffuse in neurons until they bind to MTs. Then, they walk again along the MTs to continue their tasks. Kinesins repeat this cycle of motion until they transport their cargoes to the destinations. However, most previous models mainly focused on the motion of kinesins when they walk on MTs. Thus, a new model is required to encompass the various types of kinesin motion. We developed a comprehensive model and studied the long-range axonal transport of neurons using the model. To enhance reliability of the model, it was constructed based on multiphysics on kinesin motion (i.e., chemical kinetics, diffusion, fluid dynamics, nonlinear dynamics, and stochastic characteristics). Also, parameter values for kinesin motions are carefully obtained by comparing the model predictions and several experimental observations. The axonal transport can be degraded when a large number of binding sites on MTs are blocked by excessive tau proteins. By considering the interference between walking kinesins and tau molecules on MTs, effects of tau proteins on the axonal transport are studied. One of the meaningful predictions obtained from the model is that the velocity is not an effective metric to estimate the degradation of the transport because the decrease in velocity is not noticeable when the concentration of tau protein is not high. However, our model shows that the transport locally changes near tau molecules on MTs even when the change in the velocity is not significant. Thus, a statistical method is proposed to detect this local change effectively. The advantage of this method is that a value obtained from this method is highly sensitive to the concentration of tau protein. Another benefit of this method is that this highly sensitive value can

  1. Dynamic model for kinesin-mediated long-range transport and its local traffic jam caused by tau proteins.

    PubMed

    Nam, Woochul; Epureanu, Bogdan I

    2017-01-01

    In neurons, several intracellular cargoes are transported by motor proteins (kinesins) which walk on microtubules (MTs). However, kinesins can possibly unbind from the MTs before they reach their destinations. The unbound kinesins randomly diffuse in neurons until they bind to MTs. Then, they walk again along the MTs to continue their tasks. Kinesins repeat this cycle of motion until they transport their cargoes to the destinations. However, most previous models mainly focused on the motion of kinesins when they walk on MTs. Thus, a new model is required to encompass the various types of kinesin motion. We developed a comprehensive model and studied the long-range axonal transport of neurons using the model. To enhance reliability of the model, it was constructed based on multiphysics on kinesin motion (i.e., chemical kinetics, diffusion, fluid dynamics, nonlinear dynamics, and stochastic characteristics). Also, parameter values for kinesin motions are carefully obtained by comparing the model predictions and several experimental observations. The axonal transport can be degraded when a large number of binding sites on MTs are blocked by excessive tau proteins. By considering the interference between walking kinesins and tau molecules on MTs, effects of tau proteins on the axonal transport are studied. One of the meaningful predictions obtained from the model is that the velocity is not an effective metric to estimate the degradation of the transport because the decrease in velocity is not noticeable when the concentration of tau protein is not high. However, our model shows that the transport locally changes near tau molecules on MTs even when the change in the velocity is not significant. Thus, a statistical method is proposed to detect this local change effectively. The advantage of this method is that a value obtained from this method is highly sensitive to the concentration of tau protein. Another benefit of this method is that this highly sensitive value can

  2. The heterotrimeric kinesin-2 complex interacts with and regulates GLI protein function.

    PubMed

    Carpenter, Brandon S; Barry, Renee L; Verhey, Kristen J; Allen, Benjamin L

    2015-03-01

    GLI transport to the primary cilium and nucleus is required for proper Hedgehog (HH) signaling; however, the mechanisms that mediate these trafficking events are poorly understood. Kinesin-2 motor proteins regulate ciliary transport of cargo, yet their role in GLI protein function remains unexplored. To examine a role for the heterotrimeric KIF3A-KIF3B-KAP3 kinesin-2 motor complex in regulating GLI activity, we performed a series of structure-function analyses using biochemical, cell signaling and in vivo approaches that define novel specific interactions between GLI proteins and two components of this complex, KAP3 and KIF3A. We find that all three mammalian GLI proteins interact with KAP3 and we map specific interaction sites in both proteins. Furthermore, we find that GLI proteins interact selectively with KIF3A, but not KIF3B, and that GLI interacts synergistically with KAP3 and KIF3A. Using a combination of cell signaling assays and chicken in ovo electroporation, we demonstrate that KAP3 interactions restrict GLI activator function but not GLI repressor function. These data suggest that GLI interactions with KIF3A-KIF3B-KAP3 complexes are essential for proper GLI transcriptional activity.

  3. Altered motor activity of alternative splice variants of the mammalian kinesin-3 protein KIF1B.

    PubMed

    Matsushita, Masafumi; Yamamoto, Ruri; Mitsui, Keiji; Kanazawa, Hiroshi

    2009-11-01

    Several mammalian kinesin motor proteins exist as multiple isoforms that arise from alternative splicing of a single gene. However, the roles of many motor protein splice variants remain unclear. The kinesin-3 motor protein KIF1B has alternatively spliced isoforms distinguished by the presence or absence of insertion sequences in the conserved amino-terminal region of the protein. The insertions are located in the loop region containing the lysine-rich cluster, also known as the K-loop, and in the hinge region adjacent to the motor domain. To clarify the functions of these alternative splice variants of KIF1B, we examined the biochemical properties of recombinant KIF1B with and without insertion sequences. In a microtubule-dependent ATPase assay, KIF1B variants that contained both insertions had higher activity and affinity for microtubules than KIF1B variants that contained no insertions. Mutational analysis of the K-loop insertion revealed that variants with a longer insertion sequence at this site had higher activity. However, the velocity of movement in motility assays was similar between KIF1B with and without insertion sequences. Our results indicate that splicing isoforms of KIF1B that vary in their insertion sequences have different motor activities.

  4. Vaccinia Protein F12 Has Structural Similarity to Kinesin Light Chain and Contains a Motor Binding Motif Required for Virion Export

    PubMed Central

    Morgan, Gareth W.; Hollinshead, Michael; Ferguson, Brian J.; Murphy, Brendan J.; Carpentier, David C. J.; Smith, Geoffrey L.

    2010-01-01

    Vaccinia virus (VACV) uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC), a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs). Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD) motif, which is conserved in the kinesin-1-binding sequence (KBS) of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage. PMID:20195521

  5. The 3Ms of central spindle assembly: microtubules, motors and MAPs.

    PubMed

    Glotzer, Michael

    2009-01-01

    During metaphase, sister chromatids are positioned at the midpoint of the microtubule-based mitotic spindle in preparation for their segregation. The onset of anaphase triggers inactivation of the key mitotic kinase cyclin-dependent kinase 1 (CDK1) and the polewards movement of sister chromatids. During anaphase, the mitotic spindle reorganizes in preparation for cytokinesis. Kinesin motor proteins and microtubule-associated proteins bundle the plus ends of interpolar microtubules and generate the central spindle, which regulates cleavage furrow initiation and the completion of cytokinesis. Complementary approaches, including cell biology, genetics and computational modelling, have provided new insights into the mechanism and regulation of central spindle assembly.

  6. Two kinesin-like Kin I family proteins in fission yeast regulate the establishment of metaphase and the onset of anaphase A.

    PubMed

    Garcia, Miguel Angel; Koonrugsa, Nirada; Toda, Takashi

    2002-04-16

    Metaphase is thought to be a force-equilibrium state of "tug of war," in which poleward forces are pulling kinetochores and counteracting the cohesive forces between the centromeres. Unlike conventional kinesins, members of the Kin I family are microtubule-depolymerizing enzymes, which are expected to be molecules that could generate poleward forces. We have characterized mitotic roles of two Kin I homologs, Klp5 and Klp6, in fission yeast. Klp5 and Klp6 colocalize to the mitotic kinetochores and the spindle midzone. These two proteins form a heterocomplex, but not a homocomplex. Albeit not essential, both proteins are required for accurate chromosome segregation and normal morphology of interphase microtubules. Time-lapse live analysis using GFP-alpha-tubulin indicates that these mutants spend a much longer time (2-fold) in mitosis before the initiation of anaphase B. Further observation using kinetochore and centromere markers shows that, in these mutants, sister centromeres move back and forth between the two poles, indicating that entry into anaphase A is delayed. This is supported by live image analysis showing that Cut2 securin is retained during the prolonged mitosis. Furthermore, the mitotic extension is dependent upon the Mad2 spindle checkpoint. We discuss two models of Kin I function in fission yeast. One proposes that Klp5 and Klp6 are required for efficient capturing of kinetochores by the spindles, while the other proposes that they are required to generate tension upon kinetochore capturing. Kin I, therefore, plays a fundamental role in the establishment of metaphase, probably by generating poleward forces at the kinetochores.

  7. WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression

    PubMed Central

    Bogoyevitch, Marie A.; Yeap, Yvonne Y. C.; Qu, Zhengdong; Ngoei, Kevin R.; Yip, Yan Y.; Zhao, Teresa T.; Heng, Julian I.; Ng, Dominic C. H.

    2012-01-01

    Summary The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase–anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis. PMID:22899712

  8. WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression.

    PubMed

    Bogoyevitch, Marie A; Yeap, Yvonne Y C; Qu, Zhengdong; Ngoei, Kevin R; Yip, Yan Y; Zhao, Teresa T; Heng, Julian I; Ng, Dominic C H

    2012-11-01

    The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase-anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.

  9. Arabidopsis thaliana AUCSIA-1 Regulates Auxin Biology and Physically Interacts with a Kinesin-Related Protein

    PubMed Central

    Pii, Youry; Korte, Arthur; Spena, Angelo

    2012-01-01

    Aucsia is a green plant gene family encoding 44–54 amino acids long miniproteins. The sequenced genomes of most land plants contain two Aucsia genes. RNA interference of both tomato (Solanum lycopersicum) Aucsia genes (SlAucsia-1 and SlAucsia-2) altered auxin sensitivity, auxin transport and distribution; it caused parthenocarpic development of the fruit and other auxin-related morphological changes. Here we present data showing that the Aucsia-1 gene of Arabidopsis thaliana alters, by itself, root auxin biology and that the AtAUCSIA-1 miniprotein physically interacts with a kinesin-related protein. The AtAucsia-1 gene is ubiquitously expressed, although its expression is higher in roots and inflorescences in comparison to stems and leaves. Two allelic mutants for AtAucsia-1 gene did not display visible root morphological alterations; however both basipetal and acropetal indole-3-acetic acid (IAA) root transport was reduced as compared with wild-type plants. The transcript steady state levels of the auxin efflux transporters ATP BINDING CASSETTE subfamily B (ABCB) ABCB1, ABCB4 and ABCB19 were reduced in ataucsia-1 plants. In ataucsia-1 mutant, lateral root growth showed an altered response to i) exogenous auxin, ii) an inhibitor of polar auxin transport and iii) ethylene. Overexpression of AtAucsia-1 inhibited primary root growth. In vitro and in vivo protein-protein interaction experiments showed that AtAUCSIA-1 interacts with a 185 amino acids long fragment belonging to a 2712 amino acids long protein of unknown function (At4g31570). Bioinformatics analysis indicates that the AtAUCSIA-1 interacting protein (AtAUCSIA-1IP) clusters with a group of CENP-E kinesin-related proteins. Gene ontology predictions for the two proteins are consistent with the hypothesis that the AtAUCSIA-1/AtAUCSIA-1IP complex is involved in the regulation of the cytoskeleton dynamics underlying auxin biology. PMID:22911780

  10. The Kinesin-4 Protein KIF7 Regulates Mammalian Hedgehog Signaling by Organizing the Cilia Tip Compartment

    PubMed Central

    He, Mu; Subramanian, Radhika; Bangs, Fiona; Omelchenko, Tatiana; Liem, Karel F.; Kapoor, Tarun M.; Anderson, Kathryn V.

    2014-01-01

    Mammalian Hedgehog (Hh) signal transduction requires the primary cilium, a microtubule-based organelle, and the Gli/Sufu complexes that mediate Hh signaling are enriched at cilia tips. KIF7, a kinesin-4 family protein, is a conserved regulator of the Hh signaling pathway and a human ciliopathy protein. Here we show that KIF7 localizes to cilia tips, the site of microtubule plus-ends, where it limits cilia length and controls cilia structure. Purified recombinant KIF7 binds the plus-ends of growing microtubules in vitro, where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. KIF7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of KIF7 in the mammalian Hh pathway is to control cilia architecture and to create a single cilia tip compartment where Gli/Sufu activation can be correctly regulated. PMID:24952464

  11. The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner.

    PubMed

    Bell, Kayla M; Cha, Hyo Keun; Sindelar, Charles V; Cochran, Jared C

    2017-09-01

    Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in Saccharomyces cerevisiae, can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αβ-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single "canonical" site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate ("noncanonical") sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αβ-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner

    PubMed Central

    Bell, Kayla M.; Cha, Hyo Keun; Sindelar, Charles V.; Cochran, Jared C.

    2017-01-01

    Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in Saccharomyces cerevisiae, can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αβ-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single “canonical” site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate (“noncanonical”) sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αβ-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal. PMID:28701465

  13. Roles for kinesin and myosin during cytokinesis.

    PubMed Central

    Hepler, Peter K; Valster, Aline; Molchan, Tasha; Vos, Jan W

    2002-01-01

    Cytokinesis in higher plants involves the phragmoplast, a complex cytoplasmic structure that consists of microtubules (MTs), microfilaments (MFs) and membrane elements. Both MTs and MFs are essential for cell plate formation, although it is not clear which motor proteins are involved. Some candidate processes for motor proteins include transport of Golgi vesicles to the plane of the cell plate and the spatiotemporal organization of the cytoskeletal elements in order to achieve proper deposition and alignment of the cell plate. We have focused on the kinesin-like calmodulin binding protein (KCBP) and, more broadly, on myosins. Using an antibody that constitutively activates KCBP, we find that this MT motor, which is minus-end directed, contributes to the organization of the spindle and phragmoplast MTs. It does not participate in vesicle transport; rather, because of the orientation of the phragmoplast MTs, it is supposed that plus-end kinesins fill this role. Myosins, on the other hand, based on their inhibition with 2,3-butanedione monoxime and 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine (ML-7), are associated with the process of post-mitotic spindle/phragmoplast alignment and with late lateral expansion of the cell plate. They are also not the principal motors involved in vesicle transport. PMID:12079671

  14. The kinesin-like protein TOP promotes Aurora localisation and induces mitochondrial, chloroplast and nuclear division.

    PubMed

    Yoshida, Yamato; Fujiwara, Takayuki; Imoto, Yuuta; Yoshida, Masaki; Ohnuma, Mio; Hirooka, Shunsuke; Misumi, Osami; Kuroiwa, Haruko; Kato, Shoichi; Matsunaga, Sachihiro; Kuroiwa, Tsuneyoshi

    2013-06-01

    The cell cycle usually refers to the mitotic cycle, but the cell-division cycle in the plant kingdom consists of not only nuclear but also mitochondrial and chloroplast division cycle. However, an integrated control system that initiates division of the three organelles has not been found. We report that a novel C-terminal kinesin-like protein, three-organelle division-inducing protein (TOP), controls nuclear, mitochondrial and chloroplast divisions in the red alga Cyanidioschyzon merolae. A proteomics study revealed that TOP is a member of a complex of mitochondrial-dividing (MD) and plastid-dividing (PD) machineries (MD/PD machinery complex) just prior to constriction. After TOP localizes at the MD/PD machinery complex, mitochondrial and chloroplast divisions occur and the components of the MD/PD machinery complexes are phosphorylated. Furthermore, we found that TOP downregulation impaired both mitochondrial and chloroplast divisions. MD/PD machinery complexes were formed normally at each division site but they were neither phosphorylated nor constricted in these cells. Immunofluorescence signals of Aurora kinase (AUR) were localized around the MD machinery before constriction, whereas AUR was dispersed in the cytosol by TOP downregulation, suggesting that AUR is required for the constriction. Taken together our results suggest that TOP induces phosphorylation of MD/PD machinery components to accomplish mitochondrial and chloroplast divisions prior to nuclear division, by relocalization of AUR. In addition, given the presence of TOP homologs throughout the eukaryotes, and the involvement of TOP in mitochondrial and chloroplast division may illuminate the original function of C-terminal kinesin-like proteins.

  15. Mechanical properties of spindle poles are symmetrically balanced

    PubMed Central

    Suzuki, Kazuya; Itabashi, Takeshi; Ishiwata, Shin’ichi

    2017-01-01

    The metaphase spindle is organized for accurate chromosome segregation. One of the fundamental features of the spindle across the species is its symmetrical shape; the spindle consists of two polar arrays of microtubules at both ends. Although it has been suggested that the formation of the bipolar shape requires force balance coordination by molecular motors, i.e., kinesins and dyneins, quantitative analysis for the pole mechanics has not been conducted. Here, we demonstrate that it is not only the shape but also the stiffness and microtubule density of the pairs of pole regions are symmetrically balanced in single spindles self-assembled in Xenopus egg extracts. We found that the inhibition of dynein functions dramatically reduced the stiffness and microtubule density in the pole region. By contrast, the inhibition of one of the kinesins, Eg5, which is the antagonistic motor protein of dynein, increased the value of these parameters. Moreover, the inhibition of both dynein and Eg5 recovered these parameter values to those of non-treated spindle poles. We also found that, when one pole structure was held widened with the use of two glass microneedles, the opposite pole structure spontaneously widened, resulting in the formation of the barrel-like shaped spindle. The values of stiffness and microtubule density in the manipulated pole region decreased, following the spontaneous decrement of those in the paired unmanipulated pole region. These results suggest that the spindle possesses a mechanism to dynamically maintain its symmetry in mechanical properties. PMID:28409085

  16. Mechanical properties of spindle poles are symmetrically balanced.

    PubMed

    Suzuki, Kazuya; Itabashi, Takeshi; Ishiwata, Shin'ichi

    2017-01-01

    The metaphase spindle is organized for accurate chromosome segregation. One of the fundamental features of the spindle across the species is its symmetrical shape; the spindle consists of two polar arrays of microtubules at both ends. Although it has been suggested that the formation of the bipolar shape requires force balance coordination by molecular motors, i.e., kinesins and dyneins, quantitative analysis for the pole mechanics has not been conducted. Here, we demonstrate that it is not only the shape but also the stiffness and microtubule density of the pairs of pole regions are symmetrically balanced in single spindles self-assembled in Xenopus egg extracts. We found that the inhibition of dynein functions dramatically reduced the stiffness and microtubule density in the pole region. By contrast, the inhibition of one of the kinesins, Eg5, which is the antagonistic motor protein of dynein, increased the value of these parameters. Moreover, the inhibition of both dynein and Eg5 recovered these parameter values to those of non-treated spindle poles. We also found that, when one pole structure was held widened with the use of two glass microneedles, the opposite pole structure spontaneously widened, resulting in the formation of the barrel-like shaped spindle. The values of stiffness and microtubule density in the manipulated pole region decreased, following the spontaneous decrement of those in the paired unmanipulated pole region. These results suggest that the spindle possesses a mechanism to dynamically maintain its symmetry in mechanical properties.

  17. Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

    SciTech Connect

    Delorme, Caroline; Joshi, Monika; Allingham, John S.

    2012-11-30

    Highlights: Black-Right-Pointing-Pointer The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. Black-Right-Pointing-Pointer The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. Black-Right-Pointing-Pointer The MBP-Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance, we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the 'ATP state' of the mechanochemical cycle. This site differs from the Kar3 neck-core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.

  18. Spindle alignment regulates the dynamic association of checkpoint proteins with yeast spindle pole bodies.

    PubMed

    Caydasi, Ayse Koca; Pereira, Gislene

    2009-01-01

    In many polarized cells, the accuracy of chromosome segregation depends on the correct positioning of the mitotic spindle. In budding yeast, the spindle positioning checkpoint (SPOC) delays mitotic exit when the anaphase spindle fails to extend toward the mother-daughter axis. However it remains to be established how spindle orientation is translated to SPOC components at the yeast spindle pole bodies (SPB). Here, we used photobleaching techniques to show that the dynamics with which Bub2-Bfa1 turned over at SPBs significantly increased upon SPOC activation. A version of Bfa1 that was stably associated with SPBs rendered the cells SPOC deficient without affecting other Bub2-Bfa1 functions, demonstrating the functional importance of regulating the dynamics of Bfa1 SPB association. In addition, we established that the SPOC kinase Kin4 is the major regulator of Bfa1 residence time at SPBs. We suggest that upon SPOC activation Bfa1-Bub2 spreads throughout the cytoplasm, thereby inhibiting mitotic exit.

  19. Kinesin-8 Is a Low-Force Motor Protein with a Weakly Bound Slip State

    PubMed Central

    Jannasch, Anita; Bormuth, Volker; Storch, Marko; Howard, Jonathon; Schäffer, Erik

    2013-01-01

    During the cell cycle, kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both kinesin-8 motors were much weaker compared with other kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity. PMID:23746518

  20. Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5.

    PubMed

    Ye, Anna A; Maresca, Thomas J

    2016-01-20

    Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

  1. Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation

    PubMed Central

    Lim, Nicholas R.; Yeap, Yvonne Y. C.; Ang, Ching-Seng; Williamson, Nicholas A.; Bogoyevitch, Marie A.; Quinn, Leonie M.; Ng, Dominic C. H.

    2016-01-01

    ABSTRACT Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization. PMID:26713495

  2. Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

    PubMed Central

    Biswas, Siddhartha; Blissard, Gary W.

    2016-01-01

    ABSTRACT The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with the Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni KLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation. IMPORTANCE In two key processes of the replication cycle of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are

  3. Theory of meiotic spindle assembly

    NASA Astrophysics Data System (ADS)

    Furthauer, Sebastian; Foster, Peter; Needleman, Daniel; Shelley, Michael

    2016-11-01

    The meiotic spindle is a biological structure that self assembles from the intracellular medium to separate chromosomes during meiosis. It consists of filamentous microtubule (MT) proteins that interact through the fluid in which they are suspended and via the associated molecules that orchestrate their behavior. We aim to understand how the interplay between fluid medium, MTs, and regulatory proteins allows this material to self-organize into the spindle's highly stereotyped shape. To this end we develop a continuum model that treats the spindle as an active liquid crystal with MT turnover. In this active material, molecular motors, such as dyneins which collect MT minus ends and kinesins which slide MTs past each other, generate active fluid and material stresses. Moreover nucleator proteins that are advected with and transported along MTs control the nucleation and depolymerization of MTs. This theory captures the growth process of meiotic spindles, their shapes, and the essential features of many perturbation experiments. It thus provides a framework to think about the physics of this complex biological suspension.

  4. Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization.

    PubMed

    Lemos, C L; Sampaio, P; Maiato, H; Costa, M; Omel'yanchuk, L V; Liberal, V; Sunkel, C E

    2000-07-17

    Through mutational analysis in Drosopjila we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.

  5. Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

    PubMed Central

    Cahu, Julie; Olichon, Aurelien; Hentrich, Christian; Schek, Henry; Drinjakovic, Jovana; Zhang, Cunjie; Doherty-Kirby, Amanda; Lajoie, Gilles; Surrey, Thomas

    2008-01-01

    Background Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. Methodology/Principal Findings We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. Conclusions/Significance These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein. PMID:19079595

  6. The Msd1–Wdr8–Pkl1 complex anchors microtubule minus ends to fission yeast spindle pole bodies

    PubMed Central

    Yukawa, Masashi; Ikebe, Chiho

    2015-01-01

    The minus ends of spindle microtubules are anchored to a microtubule-organizing center. The conserved Msd1/SSX2IP proteins are localized to the spindle pole body (SPB) and the centrosome in fission yeast and humans, respectively, and play a critical role in microtubule anchoring. In this paper, we show that fission yeast Msd1 forms a ternary complex with another conserved protein, Wdr8, and the minus end–directed Pkl1/kinesin-14. Individual deletion mutants displayed the identical spindle-protrusion phenotypes. Msd1 and Wdr8 were delivered by Pkl1 to mitotic SPBs, where Pkl1 was tethered through Msd1–Wdr8. The spindle-anchoring defect imposed by msd1/wdr8/pkl1 deletions was suppressed by a mutation of the plus end–directed Cut7/kinesin-5, which was shown to be mutual. Intriguingly, Pkl1 motor activity was not required for its anchoring role once targeted to the SPB. Therefore, spindle anchoring through Msd1–Wdr8–Pkl1 is crucial for balancing the Cut7/kinesin-5–mediated outward force at the SPB. Our analysis provides mechanistic insight into the spatiotemporal regulation of two opposing kinesins to ensure mitotic spindle bipolarity. PMID:25987607

  7. Impact of the Diffusion of Microtubule-Associated Protein EB1 on Kinesin Translocation in Vitro

    NASA Astrophysics Data System (ADS)

    Lopez, Benjamin; Valentine, Megan

    2014-03-01

    Using the slowly hydrolyzable GTP analog GMPCPP, we polymerize microtubules that recapitulate the end binding behavior of EB1 along their entire length, and investigate the impact of EB1 on kinesin translocation. Through direct observation of single molecules of EB1 fused to GFP, we find that EB1 diffuses along the microtubule lattice, and that the presence of taxol affects the rate of diffusion. To test whether EB1 presence and diffusion has an effect on kinesin-driven cargo transport, we observe quantum dot labeled kinesins walking on microtubules assembled with GMPCPP and taxol and coated with EB1. We find that the addition of EB1 significantly reduces kinesin speed compared to the no EB1 condition, but when microtubules stabilized by both taxol and GMPCPP are used, the speed reduction is nearly abolished. Our data suggest a new possible mechanism for the regulation of kinesin function by EB1 in which kinesin speed is directly modulated through the interference of EB1 diffusion. Our results also raise important questions about the effects of taxol on microtubule-MAP interactions.

  8. Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts.

    PubMed

    Halpin, David; Kalab, Petr; Wang, Jay; Weis, Karsten; Heald, Rebecca

    2011-12-01

    During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.

  9. An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules

    PubMed Central

    Nicholas, Matthew P.; Rao, Lu; Gennerich, Arne

    2014-01-01

    Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments. PMID:24633799

  10. An improved optical tweezers assay for measuring the force generation of single kinesin molecules.

    PubMed

    Nicholas, Matthew P; Rao, Lu; Gennerich, Arne

    2014-01-01

    Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments.

  11. Interaction of FUN14 domain containing 1, a mitochondrial outer membrane protein, with kinesin light chain 1 via the tetratricopeptide repeat domain

    PubMed Central

    Jang, Won Hee; Jeong, Young Joo; Choi, Sun Hee; Urm, Sang-Hwa; Seog, Dae-Hyun

    2017-01-01

    Kinesin 1 is a member of the kinesin superfamily proteins (KIFs) of microtubule-dependent molecular motor proteins that transport organelles and protein complexes in cells. Kinesin 1 consists of a homo- or hetero-dimer of kinesin heavy chains (KHCs), often, although not always, associated with two kinesin light chains (KLCs). KLCs are non-motor proteins that associate with many different binding proteins and cargoes, but their binding partners have not yet been fully identified. In the present study, a yeast two-hybrid system was used to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. The results of the current study revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin 1) and KIF3A (a motor subunit of kinesin 2) in the yeast two-hybrid assay. The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. Collectively, these results indicate that KLC1 may potentially compete with LC3, a key component for autophagosome formation, to interact with FUNDC1. PMID:28123706

  12. The GTPase Gem and its partner Kif9 are required for chromosome alignment, spindle length control, and mitotic progression.

    PubMed

    Andrieu, Guillaume; Quaranta, Muriel; Leprince, Corinne; Hatzoglou, Anastassia

    2012-12-01

    Within the Ras superfamily, Gem is a small GTP-binding protein that plays a role in regulating Ca(2+) channels and cytoskeletal remodeling in interphase cells. Here, we report for the first time that Gem is a spindle-associated protein and is required for proper mitotic progression. Functionally, loss of Gem leads to misaligned chromosomes and prometaphase delay. On the basis of different experimental approaches, we demonstrate that loss of Gem by RNA interference induces spindle elongation, while its enforced expression results in spindle shortening. The spindle length phenotype is generated through deregulation of spindle dynamics on Gem depletion and requires the expression of its downstream effector, the kinesin Kif9. Loss of Kif9 induces spindle abnormalities similar to those observed when Gem expression is repressed by siRNA. We further identify Kif9 as a new regulator of spindle dynamics. Kif9 depletion increases the steady-state levels of spindle α-tubulin by increasing the rate of microtubule polymerization. Overall, this study demonstrates a novel mechanism by which Gem contributes to the mitotic progression by maintaining correct spindle length through the kinesin Kif9.

  13. Tea2p Is a Kinesin-like Protein Required to Generate Polarized Growth in Fission Yeast

    PubMed Central

    Browning, Heidi; Hayles, Jacqueline; Mata, Juan; Aveline, Lauren; Nurse, Paul; McIntosh, J. Richard

    2000-01-01

    Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2+, that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Δ cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell. PMID:11018050

  14. Kinesin Light Chain 1 Suppression Impairs Human Embryonic Stem Cell Neural Differentiation and Amyloid Precursor Protein Metabolism

    PubMed Central

    Killian, Rhiannon L.; Flippin, Jessica D.; Herrera, Cheryl M.; Almenar-Queralt, Angels; Goldstein, Lawrence S. B.

    2012-01-01

    The etiology of sporadic Alzheimer disease (AD) is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aβ) and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aβ and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC) expressing reduced levels of the kinesin light chain 1 (KLC1) Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aβ, supporting the previously established connection between KLC1, Tau and Aβ. Intriguingly, KLC1-suppressed neural precursors (NPs), isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s) of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases. PMID:22272245

  15. Controlling kinesin motor proteins in nanoengineered systems through a metal-binding on/off switch.

    PubMed

    Greene, Adrienne C; Trent, Amanda M; Bachand, George D

    2008-10-15

    A significant challenge in utilizing kinesin biomolecular motors in integrated nanoscale systems is the ability to regulate motor function in vitro. Here we report a versatile mechanism for reversibly controlling the function of kinesin biomolecular motors independent of the fuel supply (ATP). Our approach relied on inhibiting conformational changes in the neck-linker region of kinesin, a process necessary for microtubule transport. We introduced a chemical switch into the neck-linker of kinesin by genetically engineering three histidine residues to create a Zn(2+)-binding site. Gliding motility of microtubules by the mutant kinesin was successfully inhibited by >/=10 microM Zn(2+), as well as other divalent metals. Motility was successfully restored by removal of Zn(2+) using a number of different chelators. Lastly, we demonstrated the robust and cyclic nature of the switch using sequential Zn(2+)/chelator additions. Overall, this approach to controlling motor function is highly advantageous as it enables control of individual classes of biomolecular motors while maintaining a consistent level of fuel for all motors in a given system or device.

  16. Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2

    PubMed Central

    Ruane, Peter T.; Gumy, Laura F.; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J.; Hoogenraad, Casper C.; Allan, Victoria J.

    2016-01-01

    Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons. PMID:27272132

  17. Kinesin-5 is a microtubule polymerase

    PubMed Central

    Chen, Yalei; Hancock, William O

    2015-01-01

    Kinesin-5 slides antiparallel microtubules during spindle assembly, and regulates the branching of growing axons. Besides the mechanical activities enabled by its tetrameric configuration, the specific motor properties of kinesin-5 that underlie its cellular function remain unclear. Here by engineering a stable kinesin-5 dimer and reconstituting microtubule dynamics in vitro, we demonstrate that kinesin-5 promotes microtubule polymerization by increasing the growth rate and decreasing the catastrophe frequency. Strikingly, microtubules growing in the presence of kinesin-5 have curved plus ends, suggesting that the motor stabilizes growing protofilaments. Single-molecule fluorescence experiments reveal that kinesin-5 remains bound to the plus ends of static microtubules for 7 s, and tracks growing microtubule plus ends in a manner dependent on its processivity. We propose that kinesin-5 pauses at microtubule plus ends and enhances polymerization by stabilizing longitudinal tubulin–tubulin interactions, and that these activities underlie the ability kinesin-5 to slide and stabilize microtubule bundles in cells. PMID:26437877

  18. Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast.

    PubMed

    Gillett, Emily S; Espelin, Christopher W; Sorger, Peter K

    2004-02-16

    Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways. Copyright The Rockefeller University Press

  19. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies.

    PubMed

    van Hemert, Martijn J; Lamers, Gerda E M; Klein, Dionne C G; Oosterkamp, Tjerk H; Steensma, H Yde; van Heusden, G Paul H

    2002-04-16

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

  20. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies

    PubMed Central

    van Hemert, Martijn J.; Lamers, Gerda E. M.; Klein, Dionne C. G.; Oosterkamp, Tjerk H.; Steensma, H. Yde; van Heusden, G. Paul H.

    2002-01-01

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of ≈10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells. PMID:11929974

  1. Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO).

    PubMed

    Alborghetti, Marcos Rodrigo; Furlan, Ariane da Silva; da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

    2013-01-01

    Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

  2. Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

    PubMed Central

    da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L.; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

    2013-01-01

    Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth. PMID:24116125

  3. Monoclonal antibodies to kinesin heavy and light chains stain vesicle- like structures, but not microtubules, in cultured cells

    PubMed Central

    1989-01-01

    Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane- bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface. PMID:2522455

  4. NtKRP, a kinesin-12 protein, regulates embryo/seed size and seed germination via involving in cell cycle progression at the G2/M transition

    PubMed Central

    Tian, Shujuan; Wu, Jingjing; Li, Fen; Zou, Jianwei; Liu, Yuwen; Zhou, Bing; Bai, Yang; Sun, Meng-Xiang

    2016-01-01

    Kinesins comprise a superfamily of microtubule-based motor proteins involved in essential processes in plant development, but few kinesins have been functionally identified during seed development. Especially, few kinesins that regulate cell division during embryogenesis have been identified. Here we report the functional characterization of NtKRP, a motor protein of the kinesin-12 family. NtKRP is predominantly expressed in embryos and embryonic roots. NtKRP RNAi lines displayed reductions in cell numbers in the meristematic zone, in embryonic root length, and in mature embryo and seed sizes. Furthermore, we also show that CDKA;1 binds to NtKRP at the consensus phosphorylation sites and that the decreased cell numbers in NtKRP-silenced embryos are due to a delay in cell division cycle at the G2/M transition. In addition, binding between the cargo-binding tail domain of NtKRP and CDKA; 1 was also determined. Our results reveal a novel molecular pathway that regulates embryo/seed development and critical role of kinesin in temporal and spatial regulation of a specific issue of embryo developmental. PMID:27779252

  5. RED, a Spindle Pole-associated Protein, Is Required for Kinetochore Localization of MAD1, Mitotic Progression, and Activation of the Spindle Assembly Checkpoint*

    PubMed Central

    Yeh, Pei-Chi; Yeh, Chang-Ching; Chen, Yi-Cheng; Juang, Yue-Li

    2012-01-01

    The spindle assembly checkpoint (SAC) is essential for ensuring the proper attachment of kinetochores to the spindle and, thus, the precise separation of paired sister chromatids during mitosis. The SAC proteins are recruited to the unattached kinetochores for activation of the SAC in prometaphase. However, it has been less studied whether activation of the SAC also requires the proteins that do not localize to the kinetochores. Here, we show that the nuclear protein RED, also called IK, a down-regulator of human leukocyte antigen (HLA) II, interacts with the human SAC protein MAD1. Two RED-interacting regions identified in MAD1 are from amino acid residues 301–340 and 439–480, designated as MAD1(301–340) and MAD1(439–480), respectively. Our observations reveal that RED is a spindle pole-associated protein that colocalizes with MAD1 at the spindle poles in metaphase and anaphase. Depletion of RED can cause a shorter mitotic timing, a failure in the kinetochore localization of MAD1 in prometaphase, and a defect in the SAC. Furthermore, the RED-interacting peptides MAD1(301–340) and MAD1(439–480), fused to enhanced green fluorescence protein, can colocalize with RED at the spindle poles in prometaphase, and their expression can abrogate the SAC. Taken together, we conclude that RED is required for kinetochore localization of MAD1, mitotic progression, and activation of the SAC. PMID:22351768

  6. Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7.

    PubMed

    Edamatsu, M

    2016-02-11

    Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubule-binding properties of this protein in vitro.

  7. A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C. elegans

    PubMed Central

    Srinivasan, Dayalan G.; Fisk, Ridgely M.; Xu, Huihong; van den Heuvel, Sander

    2003-01-01

    The Caenorhabditis elegans coiled-coil protein LIN-5 mediates several processes in cell division that depend on spindle forces, including alignment and segregation of chromosomes and positioning of the spindle. Here, we describe two closely related proteins, GPR-1 and GPR-2 (Gprotein regulator), which associate with LIN-5 in vivo and in vitro and depend on LIN-5 for localization to the spindle and cell cortex. GPR-1/GPR-2 contain a GoLoco/GPR motif that mediates interaction with GDP-bound Gαi/o. Inactivation of lin-5, gpr-1/gpr-2, or the Gαi/o genes goa-1 and gpa-16 all cause highly similar chromosome segregation and spindle positioning defects, indicating a positive role for the LIN-5 and GPR proteins in G protein signaling. The lin-5 and gpr-1/gpr-2 genes appear to act downstream of the par polarity genes in the one- and two-cell stages and downstream of the tyrosine kinase-related genes mes-1 and src-1 at the four-cell stage. Together, these results indicate that GPR-1/GPR-2 in association with LIN-5 activate G protein signaling to affect spindle force. Polarity determinants may regulate LIN-5/GPR/Gα locally to create the asymmetric forces that drive spindle movement. Results in C. elegans and other species are consistent with a novel model for receptor-independent activation of Gαi/o signaling. PMID:12730122

  8. Toucan protein is essential for the assembly of syncytial mitotic spindles in Drosophila melanogaster.

    PubMed

    Debec, A; Grammont, M; Berson, G; Dastugue, B; Sullivan, W; Couderc, J L

    2001-12-01

    The toc gene of Drosophila melanogaster encodes a 235-kD polypeptide with a coiled-coil domain, which is highly expressed during oogenesis (Grammont et al., 1997, 2000). We now report the localization of the Toucan protein during early embryonic development. The Toucan protein is present only during the syncytial stages and is associated with the nuclear envelope and the cytoskeletal structures of the syncytial embryo. In anaphase A, Toucan is concentrated at the spindle poles near the minus end of microtubules. This microtubule association is very dynamic during the nuclear cell cycle. Mutant embryos lacking the Toucan protein are blocked in a metaphase-like state. They display abnormal and nonfunctional spindles, characterized by broad poles, detachment of the centrosomes, and failure of migration of the chromosomes. These results strongly suggest that Toucan represents a factor essential for the assembly and the function of the syncytial mitotic spindles.

  9. A leukemia fusion protein attenuates the spindle checkpoint and promotes aneuploidy

    PubMed Central

    Boyapati, Anita; Yan, Ming; Peterson, Luke F.; Biggs, Joseph R.; Le Beau, Michelle M.

    2007-01-01

    The 8;21 chromosomal translocation occurs in 15% to 40% of patients with the FAB M2 subtype of acute myeloid leukemia (AML). This chromosomal abnormality fuses part of the AML1/RUNX1 gene to the ETO/MTG8 gene and generates the AML1-ETO protein. We previously identified a C-terminal truncated AML1-ETO protein (AEtr) in a mouse leukemia model. AEtr is almost identical to the AML1-ETO exon 9a isoform expressed in leukemia patients. Here, we describe a novel function of AEtr in the development of aneuploidy through spindle checkpoint attenuation. AEtr cells had a reduced mitotic index following nocodazole treatment, suggesting a failure in a subset of cells to arrest in mitosis with a functional spindle checkpoint. Additionally, primary leukemia cells and cell lines expressing AEtr were aneuploid. Moreover, AEtr cells had reduced levels of several spindle checkpoint proteins including BubR1 and securin following treatment with the spindle poison nocodazole. These results suggest that inactivation of the spindle checkpoint may contribute to the development of aneuploidy described in t(8;21) leukemia patients. PMID:17197431

  10. Fission yeast kinesin-8 controls chromosome congression independently of oscillations

    PubMed Central

    Mary, Hadrien; Fouchard, Jonathan; Gay, Guillaume; Reyes, Céline; Gauthier, Tiphaine; Gruget, Clémence; Pécréaux, Jacques; Tournier, Sylvie; Gachet, Yannick

    2015-01-01

    ABSTRACT In higher eukaryotes, efficient chromosome congression relies, among other players, on the activity of chromokinesins. Here, we provide a quantitative analysis of kinetochore oscillations and positioning in Schizosaccharomyces pombe, a model organism lacking chromokinesins. In wild-type cells, chromosomes align during prophase and, while oscillating, maintain this alignment throughout metaphase. Chromosome oscillations are dispensable both for kinetochore congression and stable kinetochore alignment during metaphase. In higher eukaryotes, kinesin-8 family members control chromosome congression by regulating their oscillations. By contrast, here, we demonstrate that fission yeast kinesin-8 controls chromosome congression by an alternative mechanism. We propose that kinesin-8 aligns chromosomes by controlling pulling forces in a length-dependent manner. A coarse-grained model of chromosome segregation implemented with a length-dependent process that controls the force at kinetochores is necessary and sufficient to mimic kinetochore alignment, and prevents the appearance of lagging chromosomes. Taken together, these data illustrate how the local action of a motor protein at kinetochores provides spatial cues within the spindle to align chromosomes and to prevent aneuploidy. PMID:26359299

  11. Klp10A, a stem cell centrosome-enriched kinesin, balances asymmetries in Drosophila male germline stem cell division.

    PubMed

    Chen, Cuie; Inaba, Mayu; Venkei, Zsolt G; Yamashita, Yukiko M

    2016-11-25

    Asymmetric stem cell division is often accompanied by stereotypical inheritance of the mother and daughter centrosomes. However, it remains unknown whether and how stem cell centrosomes are uniquely regulated and how this regulation may contribute to stem cell fate. Here we identify Klp10A, a microtubule-depolymerizing kinesin of the kinesin-13 family, as the first protein enriched in the stem cell centrosome in Drosophila male germline stem cells (GSCs). Depletion of klp10A results in abnormal elongation of the mother centrosomes in GSCs, suggesting the existence of a stem cell-specific centrosome regulation program. Concomitant with mother centrosome elongation, GSCs form asymmetric spindle, wherein the elongated mother centrosome organizes considerably larger half spindle than the other. This leads to asymmetric cell size, yielding a smaller differentiating daughter cell. We propose that klp10A functions to counteract undesirable asymmetries that may result as a by-product of achieving asymmetries essential for successful stem cell divisions.

  12. Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes

    PubMed Central

    Gaglio, Tirso; Dionne, Mary A.; Compton, Duane A.

    1997-01-01

    The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly. PMID:9281583

  13. Arabidopsis kinesin KP1 specifically interacts with VDAC3, a mitochondrial protein, and regulates respiration during seed germination at low temperature.

    PubMed

    Yang, Xue-Yong; Chen, Zi-Wei; Xu, Tao; Qu, Zhe; Pan, Xiao-Di; Qin, Xing-Hua; Ren, Dong-Tao; Liu, Guo-Qin

    2011-03-01

    The involvement of cytoskeleton-related proteins in regulating mitochondrial respiration has been revealed in mammalian cells. However, it is unclear if there is a relationship between the microtubule-based motor protein kinesin and mitochondrial respiration. In this research, we demonstrate that a plant-specific kinesin, Kinesin-like protein 1 (KP1; At KIN14 h), is involved in respiratory regulation during seed germination at a low temperature. Using in vitro biochemical methods and in vivo transgenic cell observations, we demonstrate that KP1 is able to localize to mitochondria via its tail domain (C terminus) and specifically interacts with a mitochondrial outer membrane protein, voltage-dependent anion channel 3 (VDAC3). Targeting of the KP1-tail to mitochondria is dependent on the presence of VDAC3. When grown at 4° C, KP1 dominant-negative mutants (TAILOEs) and vdac3 mutants exhibited a higher seed germination frequency. All germinating seeds of the kp1 and vdac3 mutants had increased oxygen consumption; the respiration balance between the cytochrome pathway and the alternative oxidase pathway was disrupted, and the ATP level was reduced. We conclude that the plant-specific kinesin, KP1, specifically interacts with VDAC3 on the mitochondrial outer membrane and that both KP1 and VDAC3 regulate aerobic respiration during seed germination at low temperature.

  14. Arabidopsis Kinesin KP1 Specifically Interacts with VDAC3, a Mitochondrial Protein, and Regulates Respiration during Seed Germination at Low Temperature[W][OA

    PubMed Central

    Yang, Xue-Yong; Chen, Zi-Wei; Xu, Tao; Qu, Zhe; Pan, Xiao-Di; Qin, Xing-Hua; Ren, Dong-Tao; Liu, Guo-Qin

    2011-01-01

    The involvement of cytoskeleton-related proteins in regulating mitochondrial respiration has been revealed in mammalian cells. However, it is unclear if there is a relationship between the microtubule-based motor protein kinesin and mitochondrial respiration. In this research, we demonstrate that a plant-specific kinesin, Kinesin-like protein 1 (KP1; At KIN14 h), is involved in respiratory regulation during seed germination at a low temperature. Using in vitro biochemical methods and in vivo transgenic cell observations, we demonstrate that KP1 is able to localize to mitochondria via its tail domain (C terminus) and specifically interacts with a mitochondrial outer membrane protein, voltage-dependent anion channel 3 (VDAC3). Targeting of the KP1-tail to mitochondria is dependent on the presence of VDAC3. When grown at 4°C, KP1 dominant-negative mutants (TAILOEs) and vdac3 mutants exhibited a higher seed germination frequency. All germinating seeds of the kp1 and vdac3 mutants had increased oxygen consumption; the respiration balance between the cytochrome pathway and the alternative oxidase pathway was disrupted, and the ATP level was reduced. We conclude that the plant-specific kinesin, KP1, specifically interacts with VDAC3 on the mitochondrial outer membrane and that both KP1 and VDAC3 regulate aerobic respiration during seed germination at low temperature. PMID:21406623

  15. Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization

    PubMed Central

    Lemos, Catarina L.; Sampaio, Paula; Maiato, Helder; Costa, Madalena; Omel’yanchuk, Leonid V.; Liberal, Vasco; Sunkel, Claudio E.

    2000-01-01

    Through mutational analysis in Drosophila, we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein–Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle. PMID:10899121

  16. M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis

    PubMed Central

    Abaza, Aouatef; Soleilhac, Jean-Marc; Westendorf, Joanne; Piel, Matthieu; Crevel, Isabelle; Roux, Aurelien; Pirollet, Fabienne

    2003-01-01

    Summary The human M-Phase Phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (1), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubules-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves towards the microtubule plus-end at a 0.07 μm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis. PMID:12740395

  17. Zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning

    PubMed Central

    Wolf, Benita; Diop, Fodé; Ferraris, Pauline; Wichit, Sineewanlaya; Busso, Coralie; Missé, Dorothée

    2017-01-01

    Zika virus (ZIKV) causes congenital microcephaly. Although ZIKV can impair cell cycle progression and provoke apoptosis, which probably contributes to disease aetiology through depletion of neural progenitor cells, additional cellular mechanisms may be important. Here, we investigated whether ZIKV infection alters centrosome number and spindle positioning, because such defects are thought to be at the root of inherited primary autosomal recessive microcephaly (MCPH). In addition to HeLa cells, in which centrosome number and spindle positioning can be well monitored, we analysed retinal epithelial cells (RPE-1), as well as brain-derived microglial (CHME-5) and neural progenitor (ReN) cells, using immunofluorescence. We established that ZIKV infection leads to supernumerary foci containing centriolar proteins that in some cases drive multipolar spindle assembly, as well as spindle positioning defects in HeLa, RPE-1 and CHME-5 cells, but not in ReN cells. We uncovered similar phenotypes in HeLa cells upon infection with dengue virus (DENV-2), another flavivirus that does not target brain cells and does not cause microcephaly. We conclude that infection with Flaviviridae can increase centrosome numbers and impair spindle positioning, thus potentially contributing to microcephaly in the case of Zika. PMID:28100662

  18. Detection of the quantity of kinesin and microgravity-sensitive kinesin genes in rat bone marrow stromal cells grown in a simulated microgravity environment

    NASA Astrophysics Data System (ADS)

    Ni, Chengzhi; Wang, Chunyan; Li, Yuan; Li, Yinghui; Dai, Zhongquan; Zhao, Dongming; Sun, Hongyi; Wu, Bin

    2011-06-01

    Kinesin and kinesin-like proteins (KLPs) constitute a superfamily of microtubule motor proteins found in all eukaryotic organisms. Members of the kinesin superfamily are known to play important roles in many fundamental cellular and developmental processes. To date, few published studies have reported on the effects of microgravity on kinesin expression. In this paper, we describe the expression pattern and microgravity-sensitive genes of kinesin in rat bone marrow stromal cells cultured in a ground-based rotating bioreactor. The quantity of kinesin under the clinorotation condition was examined by immunoblot analysis with anti-kinesin. Furthermore, the distribution of kinesin at various times during clinorotation was determined by dual immunostaining, using anti-kinesin monoclonal antibody or anti-β-tubulin monoclonal antibody. In terms of kinesin quantity, we found that the ratios of the amounts of clinorotated/stationary KLPs decreased from clinorotation day 5 to day 10, although it increased on days 2 and 3. Immunofluorescence analysis revealed that kinesin in the nucleus was the first to be affected by simulated microgravity, following the kinesin at the periphery that was affected at various times during clinorotation. Real-time RT-PCR analysis of kinesin mRNA expression was performed and led to the identification of 3 microgravity-sensitive kinesin genes: KIF9, KIFC1, and KIF21A. Our results suggest that kinesin has a distinct expression pattern, and the identification of microgravity-sensitive kinesin genes offers insight into fundamental cell biology.

  19. TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

    PubMed Central

    Ma, Nan; Titus, Janel; Gable, Alyssa; Ross, Jennifer L.

    2011-01-01

    Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2–Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5. PMID:21969468

  20. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    PubMed Central

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

  1. Kinetochore Localization of Spindle Checkpoint Proteins: Who Controls Whom?D⃞

    PubMed Central

    Vigneron, Suzanne; Prieto, Susana; Bernis, Cyril; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

    2004-01-01

    The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other. PMID:15269280

  2. The structural switch of nucleotide-free kinesin

    PubMed Central

    Cao, Luyan; Cantos-Fernandes, Soraya; Gigant, Benoît

    2017-01-01

    Kinesin-1 is an ATP-dependent motor protein that moves towards microtubules (+)-ends. Whereas structures of isolated ADP-kinesin and of complexes with tubulin of apo-kinesin and of ATP-like-kinesin are available, structural data on apo-kinesin-1 in the absence of tubulin are still missing, leaving the role of nucleotide release in the structural cycle unsettled. Here, we identified mutations in the kinesin nucleotide-binding P-loop motif that interfere with ADP binding. These mutations destabilize the P-loop (T87A mutant) or magnesium binding (T92V), highlighting a dual mechanism for nucleotide release. The structures of these mutants in their apo form are either isomorphous to ADP-kinesin-1 or to tubulin-bound apo-kinesin-1. Remarkably, both structures are also obtained from the nucleotide-depleted wild-type protein. Our results lead to a model in which, when detached from microtubules, apo-kinesin possibly occupies the two conformations we characterized, whereas, upon microtubule binding, ADP-kinesin converts to the tubulin-bound apo-kinesin conformation and releases ADP. This conformation is primed to bind ATP and, therefore, to run through the natural nucleotide cycle of kinesin-1. PMID:28195215

  3. An intrinsic mechanism controls reactivation of neural stem cells by spindle matrix proteins.

    PubMed

    Li, Song; Koe, Chwee Tat; Tay, Su Ting; Tan, Angie Lay Keng; Zhang, Shenli; Zhang, Yingjie; Tan, Patrick; Sung, Wing-Kin; Wang, Hongyan

    2017-07-25

    The switch between quiescence and proliferation is central for neurogenesis and its alteration is linked to neurodevelopmental disorders such as microcephaly. However, intrinsic mechanisms that reactivate Drosophila larval neural stem cells (NSCs) to exit from quiescence are not well established. Here we show that the spindle matrix complex containing Chromator (Chro) functions as a key intrinsic regulator of NSC reactivation downstream of extrinsic insulin/insulin-like growth factor signalling. Chro also prevents NSCs from ire-entering quiescence at later stages. NSC-specific in vivo profiling has dentified many downstream targets of Chro, including a temporal transcription factor Grainy head (Grh) and a neural stem cell quiescence-inducing factor Prospero (Pros). We show that spindle matrix proteins promote the expression of Grh and repress that of Pros in NSCs to govern their reactivation. Our data demonstrate that nuclear Chro critically regulates gene expression in NSCs at the transition from quiescence to proliferation.The spindle matrix proteins, including Chro, are known to regulate mitotic spindle assembly in the cytoplasm. Here the authors show that in Drosophila larval brain, Chro promotes neural stem cell (NSC) reactivation and prevents activated NSCs from entering quiescence, and that Chro carries out such a role by regulating the expression of key transcription factors in the nucleus.

  4. AIRE is a critical spindle-associated protein in embryonic stem cells.

    PubMed

    Gu, Bin; Lambert, Jean-Philippe; Cockburn, Katie; Gingras, Anne-Claude; Rossant, Janet

    2017-07-25

    Embryonic stem (ES) cells go though embryo-like cell cycles regulated by specialized molecular mechanisms. However, it is not known whether there are ES cell-specific mechanisms regulating mitotic fidelity. Here we showed that Autoimmune Regulator (Aire), a transcription coordinator involved in immune tolerance processes, is a critical spindle-associated protein in mouse ES(mES) cells. BioID analysis showed that AIRE associates with spindle-associated proteins in mES cells. Loss of function analysis revealed that Aire was important for centrosome number regulation and spindle pole integrity specifically in mES cells. We also identified the c-terminal LESLL motif as a critical motif for AIRE's mitotic function. Combined maternal and zygotic knockout further revealed Aire's critical functions for spindle assembly in preimplantation embryos. These results uncovered a previously unappreciated function for Aire and provide new insights into the biology of stem cell proliferation and potential new angles to understand fertility defects in humans carrying Aire mutations.

  5. AIRE is a critical spindle-associated protein in embryonic stem cells

    PubMed Central

    Gu, Bin; Lambert, Jean-Philippe; Cockburn, Katie; Gingras, Anne-Claude; Rossant, Janet

    2017-01-01

    Embryonic stem (ES) cells go though embryo-like cell cycles regulated by specialized molecular mechanisms. However, it is not known whether there are ES cell-specific mechanisms regulating mitotic fidelity. Here we showed that Autoimmune Regulator (Aire), a transcription coordinator involved in immune tolerance processes, is a critical spindle-associated protein in mouse ES(mES) cells. BioID analysis showed that AIRE associates with spindle-associated proteins in mES cells. Loss of function analysis revealed that Aire was important for centrosome number regulation and spindle pole integrity specifically in mES cells. We also identified the c-terminal LESLL motif as a critical motif for AIRE’s mitotic function. Combined maternal and zygotic knockout further revealed Aire’s critical functions for spindle assembly in preimplantation embryos. These results uncovered a previously unappreciated function for Aire and provide new insights into the biology of stem cell proliferation and potential new angles to understand fertility defects in humans carrying Aire mutations. DOI: http://dx.doi.org/10.7554/eLife.28131.001 PMID:28742026

  6. A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo.

    PubMed

    Encalada, Sandra E; Willis, John; Lyczak, Rebecca; Bowerman, Bruce

    2005-03-01

    During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

  7. The spindle matrix protein, Chromator, is a novel tubulin binding protein that can interact with both microtubules and free tubulin.

    PubMed

    Yao, Changfu; Wang, Chao; Li, Yeran; Ding, Yun; Rath, Uttama; Sengupta, Saheli; Girton, Jack; Johansen, Kristen M; Johansen, Jørgen

    2014-01-01

    The chromodomain protein, Chromator, is localized to chromosomes during interphase; however, during cell division together with other nuclear proteins Chromator redistributes to form a macro molecular spindle matrix complex that embeds the microtubule spindle apparatus. It has been demonstrated that the CTD of Chromator is sufficient for localization to the spindle matrix and that expression of this domain alone could partially rescue Chro mutant microtubule spindle defects. Furthermore, the presence of frayed and unstable microtubule spindles during mitosis after Chromator RNAi depletion in S2 cells indicated that Chromator may interact with microtubules. In this study using a variety of biochemical assays we have tested this hypothesis and show that Chromator not only has binding activity to microtubules with a Kd of 0.23 µM but also to free tubulin. Furthermore, we have mapped the interaction with microtubules to a relatively small stretch of 139 amino acids in the carboxy-terminal region of Chromator. This sequence is likely to contain a novel microtubule binding interface since database searches did not find any sequence matches with known microtubule binding motifs.

  8. In Vitro Assays Demonstrate That Pollen Tube Organelles Use Kinesin-Related Motor Proteins to Move along MicrotubulesW⃞

    PubMed Central

    Romagnoli, Silvia; Cai, Giampiero; Cresti, Mauro

    2003-01-01

    The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro–polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear

  9. The microtubule plus-end tracking protein ARMADILLO-REPEAT KINESIN1 promotes microtubule catastrophe in Arabidopsis.

    PubMed

    Eng, Ryan Christopher; Wasteneys, Geoffrey O

    2014-08-01

    Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1's function is redundant in cells other than those forming root hairs.

  10. F-Box Protein RcyA Controls Turnover of the Kinesin-7 Motor KipA in Aspergillus nidulans

    PubMed Central

    Herrero, Saturnino; Takeshita, Norio

    2014-01-01

    Fungal filamentous growth depends on continuous membrane insertion at the tip, the delivery of membrane-bound positional markers, and the secretion of enzymes for cell wall biosynthesis. This is achieved through exocytosis. At the same time, polarized growth requires membrane and protein recycling through endocytosis. Endocytic vesicles are thought to enter the protein degradation pathway or recycle their content to the cell surface. In Saccharomyces cerevisiae, the Rcy1 F-box protein is involved in the recycling process of a v-SNARE protein. We identified a Rcy1 orthologue, RcyA, in the filamentous fungus Aspergillus nidulans as a protein interacting with the KipA kinesin-7 motor protein in a yeast two-hybrid screen. The interaction was confirmed through bimolecular fluorescence complementation. RcyA possesses an F-box domain at the N terminus and a prenylation (CaaX) motif at the C terminus. RcyA shows also similarity to Sec10, a component of the exocyst complex. The RcyA protein localized to the hyphal tip and forming septa, likely through transportation on secretory vesicles and partially on early endosomes, but independently of KipA. Deletion of rcyA did not cause severe morphological changes but caused partial defects in the recycling of the SynA v-SNARE protein and the positioning of the cell end markers TeaA and TeaR. In addition, deletion of rcyA led to increased concentrations of the KipA protein, whereas the transcript concentration was unaffected. These results suggest that RcyA probably labels KipA for degradation and thereby controls the protein amount of KipA. PMID:24951440

  11. Unrestrained Spindle Elongation during Recovery from Spindle Checkpoint Activation in cdc15-2 Cells Results in Mis-Segregation of Chromosomes

    PubMed Central

    Chai, Chuan Chung; Teh, Ee Mei

    2010-01-01

    During normal metaphase in Saccharomyces cerevisiae, chromosomes are captured at the kinetochores by microtubules emanating from the spindle pole bodies at opposite poles of the dividing cell. The balance of forces between the cohesins holding the replicated chromosomes together and the pulling force from the microtubules at the kinetochores result in the biorientation of the sister chromatids before chromosome segregation. The absence of kinetochore–microtubule interactions or loss of cohesion between the sister chromatids triggers the spindle checkpoint which arrests cells in metaphase. We report here that an MEN mutant, cdc15-2, though competent in activating the spindle assembly checkpoint when exposed to Noc, mis-segregated chromosomes during recovery from spindle checkpoint activation. cdc15-2 cells arrested in Noc, although their Pds1p levels did not accumulate as well as in wild-type cells. Genetic analysis indicated that Pds1p levels are lower in a mad2Δ cdc15-2 and bub2Δ cdc15-2 double mutants compared with the single mutants. Chromosome mis-segregation in the mutant was due to premature spindle elongation in the presence of unattached chromosomes, likely through loss of proper control on spindle midzone protein Slk19p and kinesin protein, Cin8p. Our data indicate that a slower rate of transition through the cell division cycle can result in an inadequate level of Pds1p accumulation that can compromise recovery from spindle assembly checkpoint activation. PMID:20505077

  12. Kinesin molecular motors: Transport pathways, receptors, and human disease

    NASA Astrophysics Data System (ADS)

    Goldstein, Lawrence S. B.

    2001-06-01

    Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.

  13. The intraflagellar transport protein IFT57 is required for cilia maintenance and regulates IFT-particle-kinesin-II dissociation in vertebrate photoreceptors.

    PubMed

    Krock, Bryan L; Perkins, Brian D

    2008-06-01

    Defects in protein transport within vertebrate photoreceptors can result in photoreceptor degeneration. In developing and mature photoreceptors, proteins targeted to the outer segment are transported through the connecting cilium via the process of intraflagellar transport (IFT). In studies of vertebrate IFT, mutations in any component of the IFT particle typically abolish ciliogenesis, suggesting that IFT proteins are equally required for IFT. To determine whether photoreceptor outer segment formation depends equally on individual IFT proteins, we compared the retinal phenotypes of IFT57 and IFT88 mutant zebrafish. IFT88 mutants failed to form outer segments, whereas IFT57 mutants formed short outer segments with reduced amounts of opsin. Our phenotypic analysis revealed that IFT57 is not essential for IFT, but is required for efficient IFT. In co-immunoprecipitation experiments from whole-animal extracts, we determined that kinesin II remained associated with the IFT particle in the absence of IFT57, but IFT20 did not. Additionally, kinesin II did not exhibit ATP-dependent dissociation from the IFT particle in IFT57 mutants. We conclude that IFT20 requires IFT57 to associate with the IFT particle and that IFT57 and/or IFT20 mediate kinesin II dissociation.

  14. The kinesin-like proteins, KAC1/2, regulate actin dynamics underlying chloroplast light-avoidance in Physcomitrella patens.

    PubMed

    Shen, Zhiyuan; Liu, Yen-Chen; Bibeau, Jeffrey P; Lemoi, Kyle P; Tüzel, Erkan; Vidali, Luis

    2015-01-01

    In plants, light determines chloroplast position; these organelles show avoidance and accumulation responses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thaliana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins (called KACs) are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene-specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specific cytoskeletal elements responsible for motility, this loss-of-function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament-dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.

  15. RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

    PubMed

    Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

    2016-10-01

    Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

  16. The control of spindle length by Hsp70 and Hsp110 molecular chaperones.

    PubMed

    Makhnevych, Taras; Houry, Walid A

    2013-04-17

    Molecular chaperones are an essential group of proteins required to maintain proper protein homeostasis in the cell and include Hsp40, Hsp60, Hsp70, Hsp90, and Hsp100 among others. Hsp110 proteins form a subfamily of the Hsp70 family and seem to primarily function as nucleotide exchange factors for the Hsp70s. Data to date suggest that Hsp110 together with Hsp70 are required to ensure proper spindle assembly and nuclear distribution during cell division. More specifically, we propose that an Hsp110-Hsp70 complex modulates the activity and directionality of the kinesin-5 motor, Cin8, which is required for spindle elongation. The modulation of spindle length by molecular chaperones might be a mechanism by which cell division can be controlled especially under proteostatic stress. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

    PubMed Central

    Wu, Qindong; Sandrock, Tanya M.; Turgeon, B. Gillian; Yoder, Olen C.; Wirsel, Stefan G.; Aist, James R.

    1998-01-01

    A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology. PMID:9436993

  18. The kinesin I family member KIF5C is a novel substrate for protein kinase CK2

    SciTech Connect

    Schaefer, Barbara; Goetz, Claudia; Montenarh, Mathias

    2008-10-17

    Protein kinase CK2 is ubiquitously expressed. The holoenzyme is composed of two catalytic {alpha}- or {alpha}'-subunits and two regulatory {beta}-subunits but evidence is accumulating that the subunits can function independently. The composition of the holoenzyme as well as the expression of the individual subunits varies in different tissues, with high expression of CK2{alpha}' in testis and brain. CK2 phosphorylates a number of different substrates which are implicated in basal cellular processes such as proliferation and survival of cells. Here, we report a new substrate, KIF5C, which is a member of the kinesin 1 family of motor neuron proteins. Phosphorylation of KIF5C was demonstrated in vitro and in vivo. Using deletion mutants, a peptide library, and mutation analysis a phosphorylation site for CK2 was mapped to amino acid 338 which is located in the non-motor domain of KIF5C. Interestingly, KIF5C is phosphorylated by holoenzymes composed of CK2{alpha}/CK2{beta} and CK2{alpha}'/CK2{beta} as well as by CK2{alpha}' alone but not by CK2{alpha} alone.

  19. Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids

    PubMed Central

    Quemin, Emmanuelle R. J.; Pietilä, Maija K.; Oksanen, Hanna M.; Forterre, Patrick; Rijpstra, W. Irene C.; Schouten, Stefan; Bamford, Dennis H.; Prangishvili, David

    2015-01-01

    ABSTRACT Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and

  20. ATP-binding motifs play key roles in Krp1p, kinesin-related protein 1, function for bi-polar growth control in fission yeast

    SciTech Connect

    Rhee, Dong Keun; Cho, Bon A; Kim, Hyong Bai . E-mail: hbkim5212@hotmail.com

    2005-06-03

    Kinesin is a microtubule-based motor protein with various functions related to the cell growth and division. It has been reported that Krp1p, kinesin-related protein 1, which belongs to the kinesin heavy chain superfamily, localizes on microtubules and may play an important role in cytokinesis. However, the function of Krp1p has not been fully elucidated. In this study, we overexpressed an intact form and three different mutant forms of Krp1p in fission yeast constructed by site-directed mutagenesis in two ATP-binding motifs or by truncation of the leucine zipper-like motif (LZiP). We observed hyper-extended microtubules and the aberrant nuclear shape in Krp1p-overexpressed fission yeast. As a functional consequence, a point mutation of ATP-binding domain 1 (G89E) in Krp1p reversed the effect of Krp1p overexpression in fission yeast, whereas the specific mutation in ATP-binding domain 2 (G238E) resulted in the altered cell polarity. Additionally, truncation of the leucine zipper-like domain (LZiP) at the C-terminal of Krp1p showed a normal nuclear division. Taken together, we suggest that krp1p is involved in regulation of cell-polarized growth through ATP-binding motifs in fission yeast.

  1. Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes.

    PubMed

    Shao, Hua; Ma, Chunqi; Zhang, Xuan; Li, Ruizhen; Miller, Ann L; Bement, William M; Liu, X Johné

    2012-07-15

    Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine(122) is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.

  2. Bub3 is a spindle assembly checkpoint protein regulating chromosome segregation during mouse oocyte meiosis.

    PubMed

    Li, Mo; Li, Sen; Yuan, Ju; Wang, Zhen-Bo; Sun, Shao-Chen; Schatten, Heide; Sun, Qing-Yuan

    2009-11-02

    In mitosis, the spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes have been attached to the spindle microtubules and aligned correctly at the equatorial metaphase plate. The major checkpoint proteins in mitosis consist of mitotic arrest-deficient (Mad)1-3, budding uninhibited by benzimidazole (Bub)1, Bub3, and monopolar spindle 1(Mps1). During meiosis, for the formation of a haploid gamete, two consecutive rounds of chromosome segregation occur with only one round of DNA replication. To pull homologous chromosomes to opposite spindle poles during meiosis I, both sister kinetochores of a homologue must face toward the same pole which is very different from mitosis and meiosis II. As a core member of checkpoint proteins, the individual role of Bub3 in mammalian oocyte meiosis is unclear. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of Bub3 in mouse oocyte meiosis. Our data showed that overexpressed Bub3 inhibited meiotic metaphase-anaphase transition by preventing homologous chromosome and sister chromatid segregations in meiosis I and II, respectively. Misaligned chromosomes, abnormal polar body and double polar bodies were observed in Bub3 knock-down oocytes, causing aneuploidy. Furthermore, through cold treatment combined with Bub3 overexpression, we found that overexpressed Bub3 affected the attachments of microtubules and kinetochores during metaphase-anaphase transition. We propose that as a member of SAC, Bub3 is required for regulation of both meiosis I and II, and is potentially involved in kinetochore-microtubule attachment in mammalian oocytes.

  3. Kinesin-3 is an organelle motor in the squid giant axon.

    PubMed

    DeGiorgis, Joseph A; Petukhova, Tatyana A; Evans, Teresa A; Reese, Thomas S

    2008-11-01

    Conventional kinesin (Kinesin-1), the founding member of the kinesin family, was discovered in the squid giant axon, where it is thought to move organelles on microtubules. In this study, we identify a second squid kinesin by searching an expressed sequence tag database derived from the ganglia that give rise to the axon. The full-length open reading frame encodes a 1753 amino acid sequence that classifies this protein as a Kinesin-3. Immunoblots demonstrate that this kinesin, unlike Kinesin-1, is highly enriched in chaotropically stripped axoplasmic organelles, and immunogold electron microscopy (EM) demonstrates that Kinesin-3 is tightly bound to the surfaces of these organelles. Video microscopy shows that movements of purified organelles on microtubules are blocked, but organelles remain attached, in the presence Kinesin-3 antibody. Immunogold EM of axoplasmic spreads with antibody to Kinesin-3 decorates discrete sites on many, but not all, free organelles and localizes Kinesin-3 to organelle/microtubule interfaces. In contrast, label for Kinesin-1 decorates microtubules but not organelles. The presence of Kinesin-3 on purified organelles, the ability of an antibody to block their movements along microtubules, the tight association of Kinesin-3 with motile organelles and its distribution at the interface between native organelles and microtubules suggest that Kinesin-3 is a dominant motor in the axon for unidirectional movement of organelles along microtubules.

  4. Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

    NASA Technical Reports Server (NTRS)

    Tsai, M. Y.; Morfini, G.; Szebenyi, G.; Brady, S. T.

    2000-01-01

    The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

  5. Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

    NASA Technical Reports Server (NTRS)

    Tsai, M. Y.; Morfini, G.; Szebenyi, G.; Brady, S. T.

    2000-01-01

    The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

  6. PCTK1 Regulates Integrin-Dependent Spindle Orientation via Protein Kinase A Regulatory Subunit KAP0 and Myosin X

    PubMed Central

    Iwano, Sayaka; Satou, Ayaka; Matsumura, Shigeru; Sugiyama, Naoyuki

    2015-01-01

    Integrin-dependent cell-extracellular matrix (ECM) adhesion is a determinant of spindle orientation. However, the signaling pathways that couple integrins to spindle orientation remain elusive. Here, we show that PCTAIRE-1 kinase (PCTK1), a member of the cyclin-dependent kinases (CDKs) whose function is poorly characterized, plays an essential role in this process. PCTK1 regulates spindle orientation in a kinase-dependent manner. Phosphoproteomic analysis together with an RNA interference screen revealed that PCTK1 regulates spindle orientation through phosphorylation of Ser83 on KAP0, a regulatory subunit of protein kinase A (PKA). This phosphorylation is dispensable for KAP0 dimerization and for PKA binding but is necessary for its interaction with myosin X, a regulator of spindle orientation. KAP0 binds to the FERM domain of myosin X and enhances the association of myosin X-FERM with β1 integrin. This interaction between myosin X-FERM and β1 integrin appeared to be crucial for spindle orientation control. We propose that PCTK1-KAP0-myosin X-β1 integrin is a functional module providing a link between ECM and the actin cytoskeleton in the ECM-dependent control of spindle orientation. PMID:25605337

  7. Emerging molecular mechanisms that power and regulate the anastral mitotic spindle of flowering plants.

    PubMed

    Bannigan, Alex; Lizotte-Waniewski, Michelle; Riley, Margaret; Baskin, Tobias I

    2008-01-01

    Flowering plants, lacking centrosomes as well as dynein, assemble their mitotic spindle via a pathway that is distinct visually and molecularly from that of animals and yeast. The molecular components underlying mitotic spindle assembly and function in plants are beginning to be discovered. Here, we review recent evidence suggesting the preprophase band in plants functions analogously to the centrosome in animals in establishing spindle bipolarity, and we review recent progress characterizing the roles of specific motor proteins in plant mitosis. Loss of function of certain minus-end-directed KIN-14 motor proteins causes a broadening of the spindle pole; whereas, loss of function of a KIN-5 causes the formation of monopolar spindles, resembling those formed when the homologous motor protein (e.g., Eg5) is knocked out in animal cells. We present a phylogeny of the kinesin-5 motor domain, which shows deep divergence among plant sequences, highlighting possibilities for specialization. Finally, we review information concerning the roles of selected structural proteins at mitosis as well as recent findings concerning regulation of M-phase in plants. Insight into the mitotic spindle will be obtained through continued comparison of mitotic mechanisms in a diversity of cells.

  8. Reeling in chromosomes to spindle poles: The roles of microtubule-destabilizing enzymes in mitotic spindle dynamics

    NASA Astrophysics Data System (ADS)

    Sharp, David

    2004-03-01

    The central purpose of mitosis is achieved during anaphase when sister chromatids disjoin and translocate towards opposite poles of a microtubule-based machine termed mitotic spindle. We have identified two functionally distinct microtubule-destabilizing Kin I kinesin enzymes that are responsible for normal chromatid-to-pole motion during anaphase in Drosophila. One of them, KLP59C, is required to depolymerize MTs specifically at their kinetochore-associated "plus-ends" such that chromosomes 'chew' their way poleward. The second, KLP10A, is required to depolymerize MTs specifically at their pole-associated "minus-ends" thereby 'reeling' chromosomes into spindle poles. These findings provide the first description of the protein machinery that drives anaphase chromatid segregation by actively depolymerizing kinetochore MTs at both ends.

  9. Downregulation of Protein 4.1R impairs centrosome function,bipolar spindle organization and anaphase

    SciTech Connect

    Spence, Jeffrey R.; Go, Minjoung M.; Bahmanyar, S.; Barth,A.I.M.; Krauss, Sharon Wald

    2006-03-17

    Centrosomes nucleate and organize interphase MTs and areinstrumental in the assembly of the mitotic bipolar spindle. Here wereport that two members of the multifunctional protein 4.1 family havedistinct distributions at centrosomes. Protein 4.1R localizes to maturecentrioles whereas 4.1G is a component of the pericentriolar matrixsurrounding centrioles. To selectively probe 4.1R function, we used RNAinterference-mediated depletion of 4.1R without decreasing 4.1Gexpression. 4.1R downregulation reduces MT anchoring and organization atinterphase and impairs centrosome separation during prometaphase.Metaphase chromosomes fail to properly condense/align and spindleorganization is aberrant. Notably 4.1R depletion causes mislocalizationof its binding partner NuMA (Nuclear Mitotic Apparatus Protein),essential for spindle pole focusing, and disrupts ninein. Duringanaphase/telophase, 4.1R-depleted cells have lagging chromosomes andaberrant MT bridges. Our data provide functional evidence that 4.1R makescrucial contributions to centrosome integrity and to mitotic spindlestructure enabling mitosis and anaphase to proceed with the coordinatedprecision required to avoid pathological events.

  10. EFHC1, a protein mutated in juvenile myoclonic epilepsy, associates with the mitotic spindle through its N-terminus

    SciTech Connect

    Nijs, Laurence de; Lakaye, Bernard; Coumans, Bernard; Leon, Christine; Ikeda, Takashi; Delgado-Escueta, Antonio V.; Chanas, Grazyna . E-mail: G.Chanas@ulg.ac.be

    2006-09-10

    A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we expressed EGFP-tagged protein in various cell lines. In interphase cells, the fusion protein was present in the cytoplasm and in the nucleus with specific accumulation at the centrosome. During mitosis EGFP-EFHC1 colocalized with the mitotic spindle, especially at spindle poles and with the midbody during cytokinesis. Using a specific antibody, we demonstrated the same distribution of the endogenous protein. Deletion analyses revealed that the N-terminal region of EFHC1 is crucial for the association with the mitotic spindle and the midbody. Our results suggest that EFHC1 could play an important role during cell division.

  11. Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase.

    PubMed

    Krauss, Sharon Wald; Spence, Jeffrey R; Bahmanyar, Shirin; Barth, Angela I M; Go, Minjoung M; Czerwinski, Debra; Meyer, Adam J

    2008-04-01

    Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.

  12. Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator.

    PubMed

    Fabian, Lacramioara; Xia, Xuequin; Venkitaramani, Deepa V; Johansen, Kristen M; Johansen, Jørgen; Andrew, Deborah J; Forer, Arthur

    2007-07-01

    Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which we identified by mass spectrometry analysis as being titin. The spindle matrix proteins skeletor, megator and chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein.

  13. A role for central spindle proteins in cilia structure and function

    PubMed Central

    Smith, Katherine R.; Kieserman, Esther K.; Wang, Peggy I.; Basten, Sander G.; Giles, Rachel H.; Marcotte, Edward M.; Wallingford, John B.

    2013-01-01

    Cytokinesis and ciliogenesis are fundamental cellular processes that require strict coordination of microtubule organization and directed membrane trafficking. These processes have been intensely studied, but there has been little indication that regulatory machinery might be extensively shared between them. Here, we show that several central spindle/midbody proteins (PRC1, MKLP-1, INCENP, centriolin) also localize in specific patterns at the basal body complex in vertebrate ciliated epithelial cells. Moreover, bioinformatic comparisons of midbody and cilia proteomes reveal a highly significant degree of overlap. Finally, we used temperature-sensitive alleles of PRC1/spd-1 and MKLP-1/zen-4 in C. elegans to assess ciliary functions while bypassing these proteins' early role in cell division. These mutants displayed defects in both cilia function and cilia morphology. Together, these data suggest the conserved re-use of a surprisingly large number of proteins in the cytokinetic apparatus and in cilia. PMID:21246755

  14. Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein.

    PubMed

    Hirokawa, N; Takemura, R; Hisanaga, S

    1985-11-01

    We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three-dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button-like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule-associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75-kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic

  15. Cytoskeleton-associated protein 5 and clathrin heavy chain binding regulates spindle assembly in mouse oocytes

    PubMed Central

    Wang, Dong-Hui; Han, Zhe; Kong, Xiang-Wei; Ma, Yu-Zhen; Yun, Zhi-Zhong; Liang, Cheng-Guang

    2017-01-01

    Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to clathrin heavy chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation. PMID:28177917

  16. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4.

    PubMed

    Chan, Leon Y; Amon, Angelika

    2009-07-15

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function.

  17. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4

    PubMed Central

    Chan, Leon Y.; Amon, Angelika

    2009-01-01

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function. PMID:19605686

  18. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

    PubMed Central

    Gryaznova, Yuliya; Caydasi, Ayse Koca; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001 PMID:27159239

  19. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes.

    PubMed

    Gryaznova, Yuliya; Koca Caydasi, Ayse; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-05-09

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control.

  20. Kinesin superfamily protein 17 contributes to the development of bone cancer pain by participating in NR2B transport in the spinal cord of mice.

    PubMed

    Liu, Ming; Liu, Yue; Hou, Bailing; Bu, Dan; Shi, Linyu; Gu, Xiaoping; Ma, Zhengliang

    2015-03-01

    Τreatment of bone cancer pain remains a challenge, while the mechanisms causing the pain remain elusive. We demonstrated that the expression of the N‑methyl‑D‑aspartate (NMDA) receptor NR2B subunit was upregulated in mice with bone cancer pain. Kinesin superfamily protein 17 (KIF17), a recently characterized member of the kinesin superfamily proteins, has been demonstrated to transport and deliver the NR2B subunit to dendrites in mammalian neurons. In the present study, we induced bone cancer pain via femur bone cavity osteosarcoma NCTC 2472 tumor cell implantation (TCI) in mice. The results showed that TCI in mice increased the number of spontaneous flinches, mechanical allodynia events, expression of spinal KIF17 and NR2B subunits. Intrathecal administration of KIF17 antisense oligodeoxynucleotide (ODN) attenuated the behavioral signs of bone cancer pain and suppressed the increased expression of NR2B induced by TCI. In addition, KIF17 binds to a protein complex that contains mLin‑10 to transport NR2B, and we determined that the increase of mLin‑10 was suppressed following admini-stration. Thus, these findings suggested that KIF17 contributed to the development of bone cancer pain in the spinal cord through NR2B transport and that mLin‑10 may also play a role in pain development.

  1. Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

    PubMed

    Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

    2017-05-02

    Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

  2. Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

    PubMed Central

    Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

    2017-01-01

    Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit. DOI: http://dx.doi.org/10.7554/eLife.25366.001 PMID:28463114

  3. Construction of protein phosphorylation networks by data mining, text mining and ontology integration: analysis of the spindle checkpoint

    PubMed Central

    Ross, Karen E.; Arighi, Cecilia N.; Ren, Jia; Huang, Hongzhan; Wu, Cathy H.

    2013-01-01

    Knowledge representation of the role of phosphorylation is essential for the meaningful understanding of many biological processes. However, such a representation is challenging because proteins can exist in numerous phosphorylated forms with each one having its own characteristic protein–protein interactions (PPIs), functions and subcellular localization. In this article, we evaluate the current state of phosphorylation event curation and then present a bioinformatics framework for the annotation and representation of phosphorylated proteins and construction of phosphorylation networks that addresses some of the gaps in current curation efforts. The integrated approach involves (i) text mining guided by RLIMS-P, a tool that identifies phosphorylation-related information in scientific literature; (ii) data mining from curated PPI databases; (iii) protein form and complex representation using the Protein Ontology (PRO); (iv) functional annotation using the Gene Ontology (GO); and (v) network visualization and analysis with Cytoscape. We use this framework to study the spindle checkpoint, the process that monitors the assembly of the mitotic spindle and blocks cell cycle progression at metaphase until all chromosomes have made bipolar spindle attachments. The phosphorylation networks we construct, centered on the human checkpoint kinase BUB1B (BubR1) and its yeast counterpart MAD3, offer a unique view of the spindle checkpoint that emphasizes biologically relevant phosphorylated forms, phosphorylation-state–specific PPIs and kinase–substrate relationships. Our approach for constructing protein phosphorylation networks can be applied to any biological process that is affected by phosphorylation. Database URL: http://www.yeastgenome.org/ PMID:23749465

  4. Kif2a regulates spindle organization and cell cycle progression in meiotic oocytes

    PubMed Central

    Yi, Zi-Yun; Ma, Xue-Shan; Liang, Qiu-Xia; Zhang, Teng; Xu, Zhao-Yang; Meng, Tie-Gang; Ouyang, Ying-Chun; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan; Quan, Song

    2016-01-01

    Kif2a is a member of the Kinesin-13 microtubule depolymerases. Here, we report the expression, subcellular localization and functions of Kif2a during mouse oocyte meiotic maturation. Immunoblotting analysis showed that Kif2a was gradually increased form GV to the M I stages, and then decreased slightly at the M II stage. Confocal microscopy identified that Kif2a localized to the meiotic spindle, especially concentrated at the spindle poles and inner centromeres in metaphase and translocated to the midbody at telophase. Kif2a depletion by siRNA microinjection generated severely defective spindles and misaligned chromosomes, reduced microtubule depolymerization, which led to significant pro-M I/M Iarrest and failure of first polar body (PB1) extrusion. Kif2a-depleted oocytes were also defective in spindle pole localization of γ-tubulin and showed spindle assembly checkpoint (SAC) protein Bub3 at the kinetochores even after 10 hr extended culture. These results demonstrate that Kif2a may act as a microtubule depolymerase, regulating microtubule dynamics, spindle assembly and chromosome congression, and thus cell cycle progression during mouse oocyte meiotic maturation. PMID:27991495

  5. Small molecule screen for candidate antimalarials targeting Plasmodium Kinesin-5.

    PubMed

    Liu, Liqiong; Richard, Jessica; Kim, Sunyoung; Wojcik, Edward J

    2014-06-06

    Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable "next generation" target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are "druggable." One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease.

  6. Formin-mediated actin polymerization cooperates with Mushroom body defect (Mud)–Dynein during Frizzled–Dishevelled spindle orientation

    PubMed Central

    Johnston, Christopher A.; Manning, Laurina; Lu, Michelle S.; Golub, Ognjen; Doe, Chris Q.; Prehoda, Kenneth E.

    2013-01-01

    Summary To position the mitotic spindle, cytoskeletal components must be coordinated to generate cortical forces on astral microtubules. Although the dynein motor is common to many spindle orientation systems, ‘accessory pathways’ are often also required. In this work, we identified an accessory spindle orientation pathway in Drosophila that functions with Dynein during planar cell polarity, downstream of the Frizzled (Fz) effector Dishevelled (Dsh). Dsh contains a PDZ ligand and a Dynein-recruiting DEP domain that are both required for spindle orientation. The Dsh PDZ ligand recruits Canoe/Afadin and ultimately leads to Rho GTPase signaling mediated through RhoGEF2. The formin Diaphanous (Dia) functions as the Rho effector in this pathway, inducing F-actin enrichment at sites of cortical Dsh. Chimeric protein experiments show that the Dia–actin accessory pathway can be replaced by an independent kinesin (Khc73) accessory pathway for Dsh-mediated spindle orientation. Our results define two ‘modular’ spindle orientation pathways and show an essential role for actin regulation in Dsh-mediated spindle orientation. PMID:23868974

  7. KIF2A regulates the spindle assembly and the metaphase I-anaphase I transition in mouse oocyte

    PubMed Central

    Chen, Ming-Huang; Liu, Yu; Wang, Ya-Long; Liu, Rui; Xu, Bai-Hui; Zhang, Fei; Li, Fei-Ping; Xu, Lin; Lin, Yan-Hong; He, Shu-Wen; Liao, Bao-Qiong; Fu, Xian-Pei; Wang, Xiao-Xue; Yang, Xiang-Jun; Wang, Hai-Long

    2016-01-01

    KIF2A, a member of the kinesin-13 family, has been reported to play a role in spindle assembly in mitosis. However, its function in mammalian meiosis remains unknown. In this research, we examined the expression, localization and function of KIF2A during mouse oocyte meiosis. KIF2A was expressed in some key stages in mouse oocyte meiosis. Immunofluorescent staining showed that KIF2A distributed in the germinal vesicle at the germinal vesicle stage and as the spindle assembling after meiosis resumption, KIF2A gradually accumulated to the entire spindle. The treatment of oocytes with taxol and nocodazole demonstrated that KIF2A was co-localized with α-tubulin. Depletion of KIF2A by specific short interfering (si) RNA injection resulted in abnormal spindle assembly, failure of spindle migration, misaligned chromosomes and asymmetric cell division. Meanwhile, SKA1 expression level was decreased and the TACC3 localization was disrupted. Moreover, depletion of KIF2A disrupted the actin cap formation, arrested oocytes at metaphase I with spindle assembly checkpoint protein BubR1 activated and finally reduced the rate of the first polar body extrusion. Our data indicate that KIF2A regulates the spindle assembly, asymmetric cytokinesis and the metaphase I-anaphase I transition in mouse oocyte. PMID:27991556

  8. The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4.

    PubMed

    Bertazzi, Daniela Trinca; Kurtulmus, Bahtiyar; Pereira, Gislene

    2011-06-13

    The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2-Bfa1 GAP complex that inhibit the mitotic exit-promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2-Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2-Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.

  9. The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4

    PubMed Central

    Bertazzi, Daniela Trinca; Kurtulmus, Bahtiyar

    2011-01-01

    The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2–Bfa1 GAP complex that inhibit the mitotic exit–promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2–Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2–Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB. PMID:21670215

  10. Interplay between Velocity and Travel Distance of Kinesin-based Transport in the Presence of Tau

    PubMed Central

    Xu, Jing; King, Stephen J.; Lapierre-Landry, Maryse; Nemec, Brian

    2013-01-01

    Although the disease-relevant microtubule-associated protein tau is known to severely inhibit kinesin-based transport in vitro, the potential mechanisms for reversing this detrimental effect to maintain healthy transport in cells remain unknown. Here we report the unambiguous upregulation of multiple-kinesin travel distance despite the presence of tau, via decreased single-kinesin velocity. Interestingly, the presence of tau also modestly reduced cargo velocity in multiple-kinesin transport, and our stochastic simulations indicate that the tau-mediated reduction in single-kinesin travel underlies this observation. Taken together, our observations highlight a nontrivial interplay between velocity and travel distance for kinesin transport, and suggest that single-kinesin velocity is a promising experimental handle for tuning the effect of tau on multiple-kinesin travel distance. PMID:24268156

  11. Spindle-E cycling between nuage and cytoplasm is controlled by Qin and PIWI proteins

    PubMed Central

    Andress, Arlise; Bei, Yanxia; Fonslow, Bryan R.; Giri, Ritika; Wu, Yilong; Yates, John R.

    2016-01-01

    Transposable elements (TEs) are silenced in germ cells by a mechanism in which PIWI proteins generate and use PIWI-interacting ribonucleic acid (piRNA) to repress expression of TE genes. piRNA biogenesis occurs by an amplification cycle in microscopic organelles called nuage granules, which are localized to the outer face of the nuclear envelope. One cofactor required for amplification is the helicase Spindle-E (Spn-E). We found that the Spn-E protein physically associates with the Tudor domain protein Qin and the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3). Spn-E and Qin proteins are mutually dependent for their exit from nuage granules, whereas Spn-E and both Aub and Ago3 are mutually dependent for their entry or retention in nuage. The result is a dynamic cycling of Spn-E and its associated factors in and out of nuage granules. This implies that nuage granules can be considered to be hubs for active, mobile, and transient complexes. We suggest that this is in some way coupled with the execution of the piRNA amplification cycle. PMID:27091448

  12. A standardized kinesin nomenclature

    PubMed Central

    Lawrence, Carolyn J.; Dawe, R. Kelly; Christie, Karen R.; Cleveland, Don W.; Dawson, Scott C.; Endow, Sharyn A.; Goldstein, Lawrence S.B.; Goodson, Holly V.; Hirokawa, Nobutaka; Howard, Jonathon; Malmberg, Russell L.; McIntosh, J. Richard; Miki, Harukata; Mitchison, Timothy J.; Okada, Yasushi; Reddy, Anireddy S.N.; Saxton, William M.; Schliwa, Manfred; Scholey, Jonathan M.; Vale, Ronald D.; Walczak, Claire E.; Wordeman, Linda

    2004-01-01

    In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered. PMID:15479732

  13. HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells.

    PubMed

    Breuer, Manuel; Kolano, Agnieszka; Kwon, Mijung; Li, Chao-Chin; Tsai, Ting-Fen; Pellman, David; Brunet, Stéphane; Verlhac, Marie-Hélène

    2010-12-27

    In contrast to somatic cells, formation of acentriolar meiotic spindles relies on the organization of microtubules (MTs) and MT-organizing centers (MTOCs) into a stable bipolar structure. The underlying mechanisms are still unknown. We show that this process is impaired in hepatoma up-regulated protein (Hurp) knockout mice, which are viable but female sterile, showing defective oocyte divisions. HURP accumulates on interpolar MTs in the vicinity of chromosomes via Kinesin-5 activity. By promoting MT stability in the spindle central domain, HURP allows efficient MTOC sorting into distinct poles, providing bipolarity establishment and maintenance. Our results support a new model for meiotic spindle assembly in which HURP ensures assembly of a central MT array, which serves as a scaffold for the genesis of a robust bipolar structure supporting efficient chromosome congression. Furthermore, HURP is also required for the clustering of extra centrosomes before division, arguing for a shared molecular requirement of MTOC sorting in mammalian meiosis and cancer cell division.

  14. Spindle Assembly Checkpoint Protein Cdc20 Transcriptionally Activates Expression of Ubiquitin Carrier Protein UbcH10*

    PubMed Central

    Nath, Somsubhra; Banerjee, Taraswi; Sen, Debrup; Das, Tania; Roychoudhury, Susanta

    2011-01-01

    The spindle assembly checkpoint (SAC) ensures accurate segregation of chromosomes by monitoring kinetochore attachment of spindles during mitosis. Proper progression of mitosis depends on orderly ubiquitination and subsequent degradation of various mitotic inhibitors. At the molecular level, upon removal of SAC, Cdc20 activates E3 ubiquitin ligase anaphase-promoting complex/cyclosome that, along with E2 ubiquitin-conjugating enzyme UbcH10, executes this function. Both Cdc20 and UbcH10 are overexpressed in many cancer types and are associated with defective SAC function leading to chromosomal instability. The precise mechanism of correlated overexpression of these two proteins remains elusive. We show that Cdc20 transcriptionally up-regulates UbcH10 expression. The WD40 domain of Cdc20 is required for this activity. Physical interaction between Cdc20 and anaphase-promoting complex/cyclosome-CBP/p300 complex and its subsequent recruitment to the UBCH10 promoter are involved in this transactivation process. This transcriptional regulatory function of Cdc20 was observed to be cell cycle-specific. We hypothesize that this co-regulated overexpression of both proteins contributes to chromosomal instability. PMID:21454660

  15. Three BUB1 and BUBR1/MAD3-related spindle assembly checkpoint proteins are required for accurate mitosis in Arabidopsis.

    PubMed

    Paganelli, Laetitia; Caillaud, Marie-Cécile; Quentin, Michaël; Damiani, Isabelle; Govetto, Benjamin; Lecomte, Philippe; Karpov, Pavel A; Abad, Pierre; Chabouté, Marie-Edith; Favery, Bruno

    2015-01-01

    The spindle assembly checkpoint (SAC) is a refined surveillance mechanism which ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the spindle microtubules (MT). The SAC has been extensively studied in metazoans and yeast, but little is known about its role in plants. We identified proteins interacting with a MT-associated protein MAP65-3, which plays a critical role in organising mitotic MT arrays, and carried out a functional analysis of previously and newly identified SAC components. We show that Arabidopsis SAC proteins BUB3.1, MAD2, BUBR1/MAD3s and BRK1 interact with each other and with MAP65-3. We found that two BUBR1/MAD3s interacted specifically at centromeres. When stably expressed in Arabidopsis, BRK1 localised to the kinetochores during all stages of the mitotic cell cycle. Early in mitosis, BUB3.1 and BUBR1/MAD3.1 localise to the mitotic spindle, where MAP65-3 organises spindle MTs. A double-knockout mad3.1 mad3.2 mutant presented spindle MT abnormalities, chromosome misalignments on the metaphase plate and the production of lagging chromosomes and micronuclei during mitosis. We conclude that BRK1 and BUBR1/MAD3-related proteins play a key role in ensuring faithful chromosome segregation during mitosis and that their interaction with MAP65-3 may be important for the regulation of MT-chromosome attachment. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  16. Two Related Kinesins, klp5+ and klp6+, Foster Microtubule Disassembly and Are Required for Meiosis in Fission Yeast

    PubMed Central

    West, Robert R.; Malmstrom, Terra; Troxell, Cynthia L.; McIntosh, J. Richard

    2001-01-01

    The kinesin superfamily of microtubule motor proteins is important in many cellular processes, including mitosis and meiosis, vesicle transport, and the establishment and maintenance of cell polarity. We have characterized two related kinesins in fission yeast, klp5+ and klp6+, that are amino-terminal motors of the KIP3 subfamily. Analysis of null mutants demonstrates that neither klp5+ nor klp6+, individually or together, is essential for vegetative growth, although these mutants have altered microtubule behavior. klp5Δ and klp6Δ are resistant to high concentrations of the microtubule poison thiabendazole and have abnormally long cytoplasmic microtubules that can curl around the ends of the cell. This phenotype is greatly enhanced in the cell cycle mutant cdc25–22, leading to a bent, asymmetric cell morphology as cells elongate during cell cycle arrest. Klp5p-GFP and Klp6p-GFP both localize to cytoplasmic microtubules throughout the cell cycle and to spindles in mitosis, but their localizations are not interdependent. During the meiotic phase of the life cycle, both of these kinesins are essential. Spore viability is low in homozygous crosses of either null mutant. Heterozygous crosses of klp5Δ with klp6Δ have an intermediate viability, suggesting cooperation between these proteins in meiosis. PMID:11739790

  17. Non-catalytic motor domains enable processive movement and functional diversification of the kinesin-14 Kar3

    PubMed Central

    Mieck, Christine; Molodtsov, Maxim I; Drzewicka, Katarzyna; van der Vaart, Babet; Litos, Gabriele; Schmauss, Gerald; Vaziri, Alipasha; Westermann, Stefan

    2015-01-01

    Motor proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation. Previous studies have indicated that kinesin-14 motors are non-processive enzymes, working in the context of multi-motor ensembles that collectively organize microtubule networks. In this study, we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1. By analyzing the single-molecule properties of engineered motors, we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a ‘foothold’ that allows Kar3 to bias translocation towards the minus end. This mechanism rivals the speed and run length of conventional motors, can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo. Our findings provide an example for a non-conventional translocation mechanism and can explain how Kar3 substitutes for key functions of Dynein in the yeast nucleus. DOI: http://dx.doi.org/10.7554/eLife.04489.001 PMID:25626168

  18. PRC1 controls spindle polarization and recruitment of cytokinetic factors during monopolar cytokinesis.

    PubMed

    Shrestha, Sanjay; Wilmeth, Lori Jo; Eyer, Jarrett; Shuster, Charles B

    2012-04-01

    The central spindle is a postanaphase array of microtubules that plays an essential role in organizing the signaling machinery for cytokinesis. The model by which the central spindle organizes the cytokinetic apparatus is premised on an antiparallel arrangement of microtubules, yet cells lacking spindle bipolarity are capable of generating a distal domain of ectopic furrowing when forced into mitotic exit. Because protein regulator of cytokinesis (PRC1) and kinesin family member 4A (KIF4A) are believed to play a principal role in organizing the antiparallel midzone array, we sought to clarify their roles in monopolar cytokinesis. Although both factors localized to the distal ends of microtubules during monopolar cytokinesis, depletion of PRC1 and KIF4A displayed different phenotypes. Cells depleted of PRC1 failed to form a polarized microtubule array or ectopic furrows following mitotic exit, and recruitment of Aurora B kinase, male germ cell Rac GTPase-activating protein, and RhoA to the cortex was impaired. In contrast, KIF4A depletion impaired neither polarization nor ectopic furrowing, but it did result in elongated spindles with a diffuse distribution of cytokinetic factors. Thus, even in the absence of spindle bipolarity, PRC1 appears to be essential for polarizing parallel microtubules and concentrating the factors responsible for contractile ring assembly, whereas KIF4A is required for limiting the length of anaphase microtubules.

  19. PRC1 controls spindle polarization and recruitment of cytokinetic factors during monopolar cytokinesis

    PubMed Central

    Shrestha, Sanjay; Wilmeth, Lori Jo; Eyer, Jarrett; Shuster, Charles B.

    2012-01-01

    The central spindle is a postanaphase array of microtubules that plays an essential role in organizing the signaling machinery for cytokinesis. The model by which the central spindle organizes the cytokinetic apparatus is premised on an antiparallel arrangement of microtubules, yet cells lacking spindle bipolarity are capable of generating a distal domain of ectopic furrowing when forced into mitotic exit. Because protein regulator of cytokinesis (PRC1) and kinesin family member 4A (KIF4A) are believed to play a principal role in organizing the antiparallel midzone array, we sought to clarify their roles in monopolar cytokinesis. Although both factors localized to the distal ends of microtubules during monopolar cytokinesis, depletion of PRC1 and KIF4A displayed different phenotypes. Cells depleted of PRC1 failed to form a polarized microtubule array or ectopic furrows following mitotic exit, and recruitment of Aurora B kinase, male germ cell Rac GTPase-activating protein, and RhoA to the cortex was impaired. In contrast, KIF4A depletion impaired neither polarization nor ectopic furrowing, but it did result in elongated spindles with a diffuse distribution of cytokinetic factors. Thus, even in the absence of spindle bipolarity, PRC1 appears to be essential for polarizing parallel microtubules and concentrating the factors responsible for contractile ring assembly, whereas KIF4A is required for limiting the length of anaphase microtubules. PMID:22323288

  20. Purified Kinesin Promotes Vesicle Motility and Induces Active Sliding Between Microtubules In vitro

    NASA Astrophysics Data System (ADS)

    Urrutia, Raul; McNiven, Mark A.; Albanesi, Joseph P.; Murphy, Douglas B.; Kachar, Bechara

    1991-08-01

    We examined the ability of kinesin to support the movement of adrenal medullary chromaffin granules on microtubules in a defined in vitro system. We found that kinesin and ATP are all that is required to support efficient (33% vesicle motility) and rapid (0.4-0.6 μ m/s) translocation of secretory granule membranes on microtubules in the presence of a low-salt motility buffer. Kinesin also induced the formation of microtubule asters in this buffer, with the plus ends of microtubules located at the center of each aster. This observation indicates that kinesin is capable of promoting active sliding between microtubules toward their respective plus ends, a movement analogous to that of anaphase b in the mitotic spindle. The fact that vesicle translocation, microtubule sliding, and microtubule-dependent kinesin ATPase activities are all enhanced in low-salt buffer establishes a functional parallel between this translocator and other motility ATPases, myosin, and dynein.

  1. Spindle assembly checkpoint proteins regulate and monitor meiotic synapsis in C. elegans

    PubMed Central

    Bohr, Tisha; Nelson, Christian R.; Klee, Erin

    2015-01-01

    Homologue synapsis is required for meiotic chromosome segregation, but how synapsis is initiated between chromosomes is poorly understood. In Caenorhabditis elegans, synapsis and a checkpoint that monitors synapsis depend on pairing centers (PCs), cis-acting loci that interact with nuclear envelope proteins, such as SUN-1, to access cytoplasmic microtubules. Here, we report that spindle assembly checkpoint (SAC) components MAD-1, MAD-2, and BUB-3 are required to negatively regulate synapsis and promote the synapsis checkpoint response. Both of these roles are independent of a conserved component of the anaphase-promoting complex, indicating a unique role for these proteins in meiotic prophase. MAD-1 and MAD-2 localize to the periphery of meiotic nuclei and interact with SUN-1, suggesting a role at PCs. Consistent with this idea, MAD-1 and BUB-3 require full PC function to inhibit synapsis. We propose that SAC proteins monitor the stability of pairing, or tension, between homologues to regulate synapsis and elicit a checkpoint response. PMID:26483555

  2. How does a protein with dual mitotic spindle and extracellular matrix receptor functions affect tumor susceptibility and progression?

    PubMed Central

    Tolg, Cornelia; McCarthy, James B

    2011-01-01

    The mechanisms responsible for the oncogenic effects of the hyaluronan (HA) receptor and mitotic spindle binding protein, RHAMM, are poorly understood. On one hand, extracellular RHAMM interacts with HA and cellsurface receptors such as CD44 to coordinately activate the MAPK/ERK1,2 pathway, thus contributing to the spread and proliferation of tumor cells. On the other hand, intracellular RHAMM decorates mitotic spindles and is necessary for spindle formation and progression through G2/M and overexpression or loss of RHAMM can result in multipole spindles and chromosome missegregation. The deregulation of these intracellular functions could lead to genomic instability and fuel tumor progression. This suggests that both extracellular and intracellular RHAMM can promote tumor progression. Intracellular RHAMM can bind directly to ERK1 to form complexes with ERK2, MEK1 and ERK1,2 substrates, and we present a model whereby RHAMM's function is as a scaffold protein, controlling activation and targeting of ERK1,2 to specific substrates. PMID:21655434

  3. Mutation of Rice BC12/GDD1, Which Encodes a Kinesin-Like Protein That Binds to a GA Biosynthesis Gene Promoter, Leads to Dwarfism with Impaired Cell Elongation[W][OA

    PubMed Central

    Li, Juan; Jiang, Jiafu; Qian, Qian; Xu, Yunyuan; Zhang, Cui; Xiao, Jun; Du, Cheng; Luo, Wei; Zou, Guoxing; Chen, Mingluan; Huang, Yunqing; Feng, Yuqi; Cheng, Zhukuan; Yuan, Ming; Chong, Kang

    2011-01-01

    The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin-deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA3) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice. PMID:21325138

  4. Mutation of rice BC12/GDD1, which encodes a kinesin-like protein that binds to a GA biosynthesis gene promoter, leads to dwarfism with impaired cell elongation.

    PubMed

    Li, Juan; Jiang, Jiafu; Qian, Qian; Xu, Yunyuan; Zhang, Cui; Xiao, Jun; Du, Cheng; Luo, Wei; Zou, Guoxing; Chen, Mingluan; Huang, Yunqing; Feng, Yuqi; Cheng, Zhukuan; Yuan, Ming; Chong, Kang

    2011-02-01

    The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin-deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA₃) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice.

  5. Measuring Pushing and Braking Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Microtubules.

    PubMed

    Shimamoto, Yuta; Forth, Scott; Kapoor, Tarun M

    2015-09-28

    The proper organization of the microtubule-based mitotic spindle is proposed to depend on nanometer-sized motor proteins generating forces that scale with a micron-sized geometric feature, such as microtubule overlap length. However, it is unclear whether such regulation can be achieved by any mitotic motor protein. Here, we employ an optical-trap- and total internal reflection fluorescence (TIRF)-based assay to show that ensembles of kinesin-5, a conserved mitotic motor protein, can push apart overlapping antiparallel microtubules to generate a force whose magnitude scales with filament overlap length. We also find that kinesin-5 can produce overlap-length-dependent "brake-like" resistance against relative microtubule sliding in both parallel and antiparallel geometries, an activity that has been suggested by cell biological studies but had not been directly measured. Together, these findings, along with numerical simulations, reveal how a motor protein can function as an analog converter, "reading" simple geometric and dynamic features in cytoskeletal networks to produce regulated force outputs.

  6. Growth-associated protein 43 in differentiating peripheral nerve sheath tumors from other non-neural spindle cell neoplasms.

    PubMed

    Chen, Wei-Shen; Chen, Pei-Ling; Lu, Dongsi; Lind, Anne C; Dehner, Louis P

    2014-02-01

    The malignant peripheral nerve sheath tumor is a relatively uncommon type of soft tissue sarcoma arising from a peripheral nerve or extraneural soft tissues and showing nerve sheath differentiation. The diagnosis of malignant peripheral nerve sheath tumor is one of the most challenging tasks in surgical pathology because of its uncommon type (5-10% soft tissue sarcomas), morphologic resemblance to other spindle cell neoplasms and lack of sensitive and specific immunohistochemical markers. The pathologic diagnosis is more straightforward in the clinical setting of neurofibromatosis-1, but problems are mainly centered on the non-neurofibromatosis-1 malignant peripheral nerve sheath tumors. To date, S100 protein is the most widely applied marker in the case of a suspected malignant peripheral nerve sheath tumor, yet its suboptimal sensitivity and its expression in other spindle cell neoplasms, including spindle cell melanoma, clear-cell sarcoma, leiomyosarcoma and monophasic synovial sarcoma, add to the diagnostic conundrum. Growth-associated protein 43 (GAP43), a membrane-associated phosphoprotein expressed in neuronal growth cones and Schwann cell precursors during neural development and axonal regeneration, was applied to a set of nerve sheath and non-nerve sheath spindle cell neoplasms. The findings in this study indicate that GAP43 is expressed in malignant peripheral nerve sheath tumors (n=18/21; 86%) and demonstrates a sensitivity superior to S100 protein (n=13/21; 62%). GAP43 is also positive in neurofibromas (n=17/18; 94%), schwannomas (n=11/12; 92%) and desmoplastic melanomas (n=7/10; 70%). In contrast, it is negative in the non-desmoplastic spindle cell melanomas (n=20/22; 91%). Of the other non-neural soft tissue sarcomas, GAP43 is non-reactive in most leiomyosarcomas (n=14/16; 88%) and clear-cell sarcomas (n=8/8), and only focally positive in monophasic synovial sarcomas (n=3/7; 43%). GAP43 is seemingly a highly sensitive marker for peripheral nerve

  7. Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd

    PubMed Central

    Yun, Mikyung; Bronner, C.Eric; Park, Cheon-Gil; Cha, Sun-Shin; Park, Hee-Won; Endow, Sharyn A.

    2003-01-01

    Molecular motors undergo conformational changes to produce force and move along cytoskeletal filaments. Structural changes have been detected in kinesin motors; however, further changes are expected because previous crystal structures are in the same or closely related conformations. We report here a 2.5 Å crystal structure of the minus-end kinesin, Ncd, with the coiled-coil stalk/neck and one head rotated by ∼75° relative to the other head. The two heads are asymmetrically positioned with respect to the stalk and show asymmetry of nucleotide state: one head is fully occupied, but the other is unstably bound to ADP. Unlike previous structures, our new atomic model can be fit into cryoelectron microscopy density maps of the motor attached to microtubules, where it appears to resemble a one-head-bound motor with the stalk rotated towards the minus end. Interactions between neck and motor core residues, observed in the head that moves with the stalk, are disrupted in the other head, permitting rotation of the stalk/neck. The rotation could represent a force-producing stroke that directs the motor to the minus end. PMID:14532111

  8. Dimerization of mammalian kinesin-3 motors results in superprocessive motion

    PubMed Central

    Soppina, Virupakshi; Norris, Stephen R.; Dizaji, Aslan S.; Kortus, Matt; Veatch, Sarah; Peckham, Michelle; Verhey, Kristen J.

    2014-01-01

    The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a wide range of cellular transport activities, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We performed a comprehensive analysis of mammalian kinesin-3 motors from three different subfamilies (KIF1, KIF13, and KIF16). Using Forster resonance energy transfer microscopy in live cells, we show for the first time to our knowledge that KIF16B motors undergo cargo-mediated dimerization. The molecular mechanisms that regulate the monomer-to-dimer transition center around the neck coil (NC) segment and its ability to undergo intramolecular interactions in the monomer state versus intermolecular interactions in the dimer state. Regulation of NC dimerization is unique to the kinesin-3 family and in the case of KIF13A and KIF13B requires the release of a proline-induced kink between the NC and subsequent coiled-coil 1 segments. We show that dimerization of kinesin-3 motors results in superprocessive motion, with average run lengths of ∼10 μm, and that this property is intrinsic to the dimeric kinesin-3 motor domain. This finding opens up studies on the mechanistic basis of motor processivity. Such high processivity has not been observed for any other motor protein and suggests that kinesin-3 motors are evolutionarily adapted to serve as the marathon runners of the cellular world. PMID:24706892

  9. Bead movement by single kinesin molecules studied with optical tweezers

    NASA Astrophysics Data System (ADS)

    Block, Steven M.; Goldstein, Lawrence S. B.; Schnapp, Bruce J.

    1990-11-01

    KINESIN, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility1-6. Here, microscopic silica beads7 were precoated with carrier protein8,9, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap10-12 ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at the lowest kinesin concentrations (corresponding to ~1 molecule per bead). Beads incubated with excess kinesin moved smoothly along a microtubule for many micrometres, but beads carrying from 0.17-3 kinesin molecules per bead, moved, on average, only about 1.4 µm and then spontaneously released from the microtuble. Application of the optical trap directly behind such moving beads often pulled them off the microtubule and back into the centre of the trap. This did not occur when a bead was bound by an AMP.PNP-induced rigor linkage, or when beads were propelled by several kinesin molecules. Our results are consistent with a model in which kinesin detaches briefly from the microtubule during a part of each mechanochemical cycle, rather than a model in which kinesin remains bound at all times.

  10. Dual mechanisms regulate the recruitment of spindle assembly checkpoint proteins to the budding yeast kinetochore

    PubMed Central

    Aravamudhan, Pavithra; Chen, Renjie; Roy, Babhrubahan; Sim, Janice; Joglekar, Ajit P.

    2016-01-01

    Recruitment of spindle assembly checkpoint (SAC) proteins by an unattached kinetochore leads to SAC activation. This recruitment is licensed by the Mps1 kinase, which phosphorylates the kinetochore protein Spc105 at one or more of its six MELT repeats. Spc105 then recruits the Bub3-Bub1 and Mad1-Mad2 complexes, which produce the inhibitory signal that arrests cell division. The strength of this signal depends, in part, on the number of Bub3-Bub1 and Mad1-Mad2 molecules that Spc105 recruits. Therefore regulation of this recruitment will influence SAC signaling. To understand this regulation, we established the physiological binding curves that describe the binding of Bub3-Bub1 and Mad1-Mad2 to the budding yeast kinetochore. We find that the binding of both follows the mass action law. Mps1 likely phosphorylates all six MELT repeats of Spc105. However, two mechanisms prevent Spc105 from recruiting six Bub3-Bub1 molecules: low Bub1 abundance and hindrance in the binding of more than one Bub3-Bub1 molecule to the same Spc105. Surprisingly, the kinetochore recruits two Mad1-Mad2 heterotetramers for every Bub3-Bub1 molecule. Finally, at least three MELT repeats per Spc105 are needed for accurate chromosome segregation. These data reveal that kinetochore-intrinsic and -extrinsic mechanisms influence the physiological operation of SAC signaling, potentially to maximize chromosome segregation accuracy. PMID:27170178

  11. Control of Mitotic Spindle Position by the Saccharomyces cerevisiae Formin Bni1p

    PubMed Central

    Lee, Laifong; Klee, Saskia K.; Evangelista, Marie; Boone, Charles; Pellman, David

    1999-01-01

    Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Δ cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Δ cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation. PMID:10085293

  12. Kinesin-14 is Important for Chromosome Segregation During Mitosis and Meiosis in the Ciliate Tetrahymena thermophila.

    PubMed

    Kushida, Yasuharu; Takaine, Masak; Nakano, Kentaro; Sugai, Toshiro; Vasudevan, Krishna Kumar; Guha, Mayukh; Jiang, Yu-Yang; Gaertig, Jacek; Numata, Osamu

    2017-05-01

    Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

  13. Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death.

    PubMed

    Bougé, Anne-Laure; Parmentier, Marie-Laure

    2016-03-01

    In neurodegenerative diseases such as Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT)-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau) protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc), as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases.

  14. Aurora-A and ch-TOG act in a common pathway in control of spindle pole integrity.

    PubMed

    De Luca, M; Brunetto, L; Asteriti, I A; Giubettini, M; Lavia, P; Guarguaglini, G

    2008-11-20

    Mitotic spindle assembly is a highly regulated process, crucial to ensure the correct segregation of duplicated chromosomes in daughter cells and to avoid aneuploidy, a common feature of tumors. Among the most important spindle regulators is Aurora-A, a mitotic centrosomal kinase frequently overexpressed in tumors. Here, we investigated the role of Aurora-A in spindle pole organization in human cells. We show that RNA interference-mediated Aurora-A inactivation causes pericentriolar material fragmentation in prometaphase, yielding the formation of spindles with supernumerary poles. This fragmentation does not necessarily involve centrioles and requires microtubules (MTs). Aurora-A-depleted prometaphases mislocalize the MT-stabilizing protein colonic hepatic tumor-overexpressed gene (ch-TOG), which abnormally accumulates at spindle poles, as well as the mitotic centromere-associated kinesin (MCAK), the major functional antagonist of ch-TOG, which delocalizes from poles. ch-TOG is required for extrapole formation in prometaphases lacking Aurora-A, because co-depletion of Aurora-A and ch-TOG mitigates the fragmented pole phenotype. These results indicate a novel function of Aurora-A, the regulation of ch-TOG and MCAK localization, and highlight a common pathway involving the three factors in control of spindle pole integrity.

  15. Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast

    PubMed Central

    Blackwell, Robert; Edelmaier, Christopher; Sweezy-Schindler, Oliver; Lamson, Adam; Gergely, Zachary R.; O’Toole, Eileen; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Glaser, Matthew A.; Betterton, Meredith D.

    2017-01-01

    Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and cross-linkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. We describe a physical model that exhibits de novo bipolar spindle formation. We began with physical properties of fission-yeast spindle pole body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive cross-linkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective self-assembly. By varying the features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive cross-linkers alone. We also identify characteristic failed states of spindle assembly—the persistent monopole, X spindle, separated asters, and short spindle, which are avoided by the creation and maintenance of antiparallel microtubule overlaps. Our model can guide the identification of new, multifaceted strategies to induce mitotic catastrophes; these would constitute novel strategies for cancer chemotherapy. PMID:28116355

  16. Spindle assembly checkpoint and its regulators in meiosis.

    PubMed

    Sun, Shao-Chen; Kim, Nam-Hyung

    2012-01-01

    BACKGROUND Meiosis is a unique form of cell division in which cells divide twice but DNA is duplicated only once. Errors in chromosome segregation during meiosis will result in aneuploidy, followed by loss of the conceptus during pregnancy or birth defects. During mitosis, cells utilize a mechanism called the spindle assembly checkpoint (SAC) to ensure faithful chromosome segregation. A similar mechanism has been uncovered for meiosis in the last decade, especially in the past several years. METHODS For this review, we included data and relevant information obtained through a PubMed database search for all articles published in English from 1991 through 2011 which included the term 'meiosis', 'spindle assembly checkpoint', or 'SAC'. RESULTS There are 91 studies included. Evidence for the existence of SAC functions in meiosis is provided by studies on the SAC proteins mitotic-arrest deficient-1 (Mad1), Mad2, budding uninhibited by benzimidazole-1 (Bub1), Bub3, BubR1 and Mps1; microtubule-kinetochore attachment regulators Ndc80 complex, chromosomal passenger complex, mitotic centromere-associated kinesin (MCAK), kinetochore null 1 (KNL1) and Mis12 complex and spindle stability regulators. CONCLUSIONS SAC and its regulators exist and function in meiosis, and their malfunctions may cause germ cell aneuploidy. However, species and sexual differences exist. Moreover, interaction of SAC components with other regulators is still poorly understood, which needs further study.

  17. Proteins related to the spindle and checkpoint mitotic emphasize the different pathogenesis of hypoplastic MDS.

    PubMed

    Heredia, Fabiola Fernandes; de Sousa, Juliana Cordeiro; Ribeiro Junior, Howard Lopes; Carvalho, Alex Fiorini; Magalhaes, Silvia Maria Meira; Pinheiro, Ronald Feitosa

    2014-02-01

    Some studies show that alterations in expression of proteins related to mitotic spindle (AURORAS KINASE A and B) and mitotic checkpoint (CDC20 and MAD2L1) are involved in chromosomal instability and tumor progression in various solid and hematologic malignancies. This study aimed to evaluate these genes in MDS patients. The cytogenetics analysis was carried out by G-banding, AURKA and AURKB amplification was performed using FISH, and AURKA, AURKB, CDC20 and MAD2L1 gene expression was performed by qRT-PCR in 61 samples of bone marrow from MDS patients. AURKA gene amplification was observed in 10% of the cases, which also showed higher expression levels than the control group (p=0.038). Patients with normo/hypercellular BM presented significantly higher expression levels than hypocellular BM patients, but normo and hypercellular BM groups did not differ. After logistic regression analysis, our results showed that HIGH expression levels were associated with increased risk of developing normo/hypercellular MDS. It also indicated that age is associated with AURKA, CDC20 and MAD2L1 HIGH expression levels. The distinct expression of hypocellular patients emphasizes the prognostic importance of cellularity to MDS. The amplification/high expression of AURKA suggests that the increased expression of this gene may be related to the pathogenesis of disease.

  18. Klp10A, a stem cell centrosome-enriched kinesin, balances asymmetries in Drosophila male germline stem cell division

    PubMed Central

    Chen, Cuie; Inaba, Mayu; Venkei, Zsolt G; Yamashita, Yukiko M

    2016-01-01

    Asymmetric stem cell division is often accompanied by stereotypical inheritance of the mother and daughter centrosomes. However, it remains unknown whether and how stem cell centrosomes are uniquely regulated and how this regulation may contribute to stem cell fate. Here we identify Klp10A, a microtubule-depolymerizing kinesin of the kinesin-13 family, as the first protein enriched in the stem cell centrosome in Drosophila male germline stem cells (GSCs). Depletion of klp10A results in abnormal elongation of the mother centrosomes in GSCs, suggesting the existence of a stem cell-specific centrosome regulation program. Concomitant with mother centrosome elongation, GSCs form asymmetric spindle, wherein the elongated mother centrosome organizes considerably larger half spindle than the other. This leads to asymmetric cell size, yielding a smaller differentiating daughter cell. We propose that klp10A functions to counteract undesirable asymmetries that may result as a by-product of achieving asymmetries essential for successful stem cell divisions. DOI: http://dx.doi.org/10.7554/eLife.20977.001 PMID:27885983

  19. Plant Intracellular Transport: Tracing Functions of the Retrograde Kinesin.

    PubMed

    Müller, Sabine

    2015-09-21

    Adding to its varied repertoire of functions in cell morphogenesis and cell division, a molecular motor protein of the kinesin-14 class has recently been implicated in rapid retrograde transport along cellular tracks in moss.

  20. Small molecule inhibitor of formin homology 2 domains (SMIFH2) reveals the roles of the formin family of proteins in spindle assembly and asymmetric division in mouse oocytes.

    PubMed

    Kim, Hak-Cheol; Jo, Yu-Jin; Kim, Nam-Hyung; Namgoong, Suk

    2015-01-01

    Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.

  1. The assembly of kinesin-based nanotransport systems

    NASA Astrophysics Data System (ADS)

    Oliveira, D.; Kim, D.-M.; Umetsu, M.; Kumagai, I.; Adschiri, T.; Teizer, W.

    2012-12-01

    At the nano-scale many proteins act as biological actuators for rotation or translation. Among these proteins, the building blocks of self-assembled, highly efficient natural motors, kinesin is considered a promising tool in the development of synthetic nanorobots. Conversion of chemical energy into mechanical work, harnessed by the hydrolysis of adenosine triphosphate, propels kinesin along a cytoplasmic system of fibers, known as a microtubule. Even though recent efforts were made to engineer tailor-made artificial nanotransport systems using kinesin, no systematic study investigated how these systems can be organized from the bottom up using the surface plasmon resonance technique. Here, we show that it is possible to quantitatively evaluate how each component of such nanoscopic machines is sequentially assembled by monitoring the individual association of its components, focusing specifically on the kinesin association to microtubules as well as the cargo-kinesin association. Furthermore, the kinetic parameters reported here for the microtubules and recombinant biotinylated kinesin binding process properties are of utmost importance due to the current widespread use of biotinylated kinesin in the construction of synthetic nano-machines.

  2. Structure of an intermediate conformer of the spindle checkpoint protein Mad2

    DOE PAGES

    Hara, Mayuko; Özkan, Engin; Sun, Hongbin; ...

    2015-08-24

    The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. In this paper, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2.more » Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Finally, our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.« less

  3. Structure of an intermediate conformer of the spindle checkpoint protein Mad2

    SciTech Connect

    Hara, Mayuko; Özkan, Engin; Sun, Hongbin; Yu, Hongtao; Luo, Xuelian

    2015-08-24

    The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. In this paper, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Finally, our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.

  4. Structure of an intermediate conformer of the spindle checkpoint protein Mad2

    PubMed Central

    Hara, Mayuko; Özkan, Engin; Sun, Hongbin; Yu, Hongtao; Luo, Xuelian

    2015-01-01

    The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2. PMID:26305957

  5. Growth-associated protein-43 immunohistochemical and ultrastructural changes in jaw muscle spindles of the rat following loss of occlusion.

    PubMed

    Santiwong, Peerapong; Muramoto, Takeshi; Soma, Kunimichi; Takano, Yoshiro

    2002-03-01

    The effects of complete loss of occlusion on the structural and functional status of these muscle spindles were investigated by immunohistochemistry either for protein gene product 9.5 (PGP 9.5) or growth-associated protein-43 (GAP-43) by light and electron microscopy. All the upper molars of 4-week-old Wistar rats were extracted and the erupted portions of the upper and lower incisors of the same animals were cut-off at the level of the interdental papilla every other day. In a control group, immunoreactivity for GAP-43 was positive in the developing annulospiral endings of 2-week-old rats, but was not detected in any of the muscle spindles after 3 weeks of age. At 4 weeks of age, the PGP 9.5 immunostained spindles had well-differentiated annulospiral endings. Ultrastructurally, these afferent endings showed lenticular or circular profiles in cross-sections, and were differentially indented into the intrafusal-fibres. The inner surfaces of the terminals formed rather smooth myoneural junctions, while the outer surfaces were covered only by basal lamina continuous with that of the underlying intrafusal muscle fibres. After the experimental elimination of occlusal contact, GAP-43 immunoreactivity reappeared in some nerve endings of muscle spindles by 3 days, and persisted for at least 28 days. During this period, the afferent-terminals exhibited various fine structural abnormalities such as irregular outlines and invaginated neuromuscular interfaces. Some sensory-terminal (ST) profiles were completely engulfed by intrafusal-fibres. However, GAP-43 expression and ultrastructural alterations became undetectable within a week of the end of incisal cutting and the recovery of incisal-contact. These data indicate that remodelling of nerve terminals in muscle spindles, as assessed by GAP-43 expression and ultrastructural changes, occurs soon after a loss of occlusion, and ceases if incisal-contact is restored. It is concluded that possible changes in jaw muscle function

  6. Heterotrimeric kinesin-2, together with kinesin-1, steers vesicular acetylcholinesterase movements toward the synapse.

    PubMed

    Kulkarni, Anuttama; Khan, Yasmin; Ray, Krishanu

    2017-03-01

    Acetylcholinesterase (AChE), which is implicated in the pathophysiology of neurological disorders, is distributed along the axon and enriched at the presynaptic basal lamina. It hydrolyses the neurotransmitter acetylcholine, which inhibits synaptic transmission. Aberrant AChE activity and ectopic axonal accumulation of the enzyme are associated with neurodegenerative disorders, such as Alzheimer's disease. The molecular mechanism that underlies AChE transport is still unclear. Here, we show that expression of Drosophila AChE tagged with photoactivatable green fluorescent protein and m-Cherry (GPAC) in cholinergic neurons compensates for the RNA interference-mediated knockdown of endogenous AChE activity. GPAC-AChE, which is enriched in the neuropil region of the brain, moves in the apparently vesicular form in axons with an anterograde bias in Drosophila larvae. Two anterograde motors, kinesin-1 and -2, propel distinct aspects of GPAC-AChE movements. Total loss of kinesin-2 reduces the density of anterograde traffic and increases bidirectional movements of GPAC-AChE vesicles without altering their speed. A partial loss of kinesin-1 reduces both the density and speed of anterograde GPAC-AChE traffic and enhances the pool of stationary vesicles. Together, these results suggest that combining activity of a relatively weak kinesin-2 with that of a stronger kinesin-1 motor could steer AChE-containing vesicles toward synapse, and provides a molecular basis for the observed subcellular distribution of the enzyme.-Kulkarni, A., Khan, Y., Ray, K. Heterotrimeric kinesin-2, together with kinesin-1, steers vesicular acetylcholinesterase movements toward the synapse. © FASEB.

  7. Mechanisms of Mitotic Spindle Assembly.

    PubMed

    Petry, Sabine

    2016-06-02

    Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.

  8. Mechanisms of Mitotic Spindle Assembly

    PubMed Central

    Petry, Sabine

    2016-01-01

    Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ~200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes. PMID:27145846

  9. Ubiquitin ligase RNF20/40 facilitates spindle assembly and promotes breast carcinogenesis through stabilizing motor protein Eg5

    PubMed Central

    Duan, Yang; Huo, Dawei; Gao, Jie; Wu, Heng; Ye, Zheng; Liu, Zhe; Zhang, Kai; Shan, Lin; Zhou, Xing; Wang, Yue; Su, Dongxue; Ding, Xiang; Shi, Lei; Wang, Yan; Shang, Yongfeng; Xuan, Chenghao

    2016-01-01

    Whether transcriptional regulators are functionally involved in mitosis is a fundamental question in cell biology. Here we report that the RNF20/40 complex, a major ubiquitin ligase catalysing histone H2B monoubiquitination, interacts with the motor protein Eg5 during mitosis and participates in spindle assembly. We show that the RNF20/40 complex monoubiquitinates and stabilizes Eg5. Loss of RNF20/40 results in spindle assembly defects, cell cycle arrest and apoptosis. Consistently, depletion of either RNF20/40 or Eg5 suppresses breast cancer in vivo. Significantly, RNF20/40 and Eg5 are concurrently upregulated in human breast carcinomas and high Eg5 expression is associated with poorer overall survival of patients with luminal A, or B, breast cancer. Our study uncovers an important spindle assembly role of the RNF20/40 complex, and implicates the RNF20/40-Eg5 axis in breast carcinogenesis, supporting the pursuit of these proteins as potential targets for breast cancer therapeutic interventions. PMID:27557628

  10. Novel Dendritic Kinesin Sorting Identified by Different Process Targeting of Two Related Kinesins: KIF21A and KIF21B

    PubMed Central

    Marszalek, Joseph R.; Weiner, Joshua A.; Farlow, Samuel J.; Chun, Jerold; Goldstein, Lawrence S.B.

    1999-01-01

    Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body. PMID:10225949

  11. Corticotropin-releasing hormone stimulates mitotic kinesin-like protein 1 expression via a PLC/PKC-dependent signaling pathway in hippocampal neurons.

    PubMed

    Sheng, Hui; Xu, Yongjun; Chen, Yanming; Zhang, Yanmin; Ni, Xin

    2012-10-15

    Corticotropin-releasing hormone (CRH) has been shown to modulate dendritic development in hippocampus. Mitotic kinesin-like protein 1 (MKLP1) plays key roles in dendritic differentiation. In the present study, we examined the effects of CRH on MKLP1 expression in cultured hippocampal neurons and determine subsequent signaling pathways involved. CRH dose-dependently increased MKLP1 mRNA and protein expression. This effect can be reversed by CRHR1 antagonist but not by CRHR2 antagonist. CRHR1 knockdown impaired this effect of CRH. CRH stimulated GTP-bound Gαs protein and phosphorylated phospholipase C (PLC)-β3 expression, which were blocked by CRHR1 antagonist. Transfection of GP antagonist-2A, an inhibitory peptide of Gαq protein, blocked CRH-induced phosphorylated PLC-β3 expression. PLC and PKC inhibitors completely blocked whereas adenylyl cyclase (AC) and PKA inhibitors did not affect CRH-induced MKLP1 expression. Our results indicate that CRH act on CRHR1 to induce MKLP1 expression via PLC/PKC signaling pathway. CRH may regulate MKLP1 expression, thereby modulating dendritic development.

  12. Translocation of phospho-protein kinase Cs implies their roles in meiotic-spindle organization, polar-body emission and nuclear activity in mouse eggs.

    PubMed

    Zheng, Zhen-Yu; Li, Qing-Zhang; Chen, Da-Yuan; Schatten, Heide; Sun, Qing-Yuan

    2005-02-01

    The protein kinase Cs (PKCs) are a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The phosphorylation of PKC in germ cells is not well defined. In this study, we described the subcellular localization of phopho-PKC in the process of mouse oocyte maturation, fertilization, and early embryonic mitosis. Confocal microscopy revealed that phospho-PKC (pan) was distributed abundantly in the nucleus at the germinal vesicle stage. After germinal vesicle breakdown, phospho-PKC was localized in the vicinity of the condensed chromosomes, distributed in the whole meiotic spindle, and concentrated at the spindle poles. After metaphase I, phospho-PKC was translocated gradually to the spindle mid-zone during emission of the first polar body. After sperm penetration and electrical activation, the distribution of phospho-PKC was moved from the spindle poles to the spindle mid-zone. After the extrusion of the second polar body (PB2) phospho-PKC was localized in the area between the oocyte and the PB2. In fertilized eggs, phospho-PKC was concentrated in the pronuclei except for the nucleolus. Phospho-PKC was dispersed after pronuclear envelope breakdown, but distributed on the entire spindle at mitotic metaphase. The results suggest that PKC activation may play important roles in regulating spindle organization and stabilization, polar-body extrusion, and nuclear activity during mouse oocyte meiosis, fertilization, and early embryonic mitosis.

  13. Microtubule organization by the antagonistic mitotic motors kinesin-5 and kinesin-14

    PubMed Central

    Hentrich, Christian

    2010-01-01

    During cell division, different molecular motors act synergistically to rearrange microtubules. Minus end–directed motors are thought to have a dual role: focusing microtubule ends to poles and establishing together with plus end–directed motors a balance of force between antiparallel microtubules in the spindle. We study here the competing action of Xenopus laevis kinesin-14 and -5 in vitro in situations in which these motors with opposite directionality cross-link and slide microtubules. We find that full-length kinesin-14 can form microtubule asters without additional factors, whereas kinesin-5 does not, likely reflecting an adaptation to mitotic function. A stable balance of force is not established between two antiparallel microtubules with these motors. Instead, directional instability is generated, promoting efficient motor and microtubule sorting. A nonmotor microtubule cross-linker can suppress directional instability but also impedes microtubule sorting, illustrating a conflict between stability and dynamicity of organization. These results establish the basic organizational properties of these antagonistic mitotic motors and a microtubule bundler. PMID:20439998

  14. The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5*

    PubMed Central

    Goulet, Adeline; Behnke-Parks, William M.; Sindelar, Charles V.; Major, Jennifer; Rosenfeld, Steven S.; Moores, Carolyn A.

    2012-01-01

    Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor's properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation. PMID:23135273

  15. Kinesin light chains are essential for axonal transport in Drosophila.

    PubMed

    Gindhart, J G; Desai, C J; Beushausen, S; Zinn, K; Goldstein, L S

    1998-04-20

    Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075-1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (. Cell 64:1093-1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075-1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.

  16. Microtubule acetylation promotes kinesin-1 binding and transport.

    PubMed

    Reed, Nathan A; Cai, Dawen; Blasius, T Lynne; Jih, Gloria T; Meyhofer, Edgar; Gaertig, Jacek; Verhey, Kristen J

    2006-11-07

    Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.

  17. Pten regulates spindle pole movement through Dlg1-mediated recruitment of Eg5 to centrosomes

    PubMed Central

    van Ree, Janine H.; Nam, Hyun-Ja; Jeganathan, Karthik B.; Kanakkanthara, Arun; van Deursen, Jan M.

    2016-01-01

    Phosphatase and tensin homologue (Pten) suppresses neoplastic growth by negatively regulating PI(3)K signalling through its phosphatase activity1. To gain insight into the actions of non-catalytic Pten domains in normal physiological processes and tumorigenesis2,3, we engineered mice lacking the PDZ-binding domain (PDZ-BD). Here, we show that the PDZ-BD regulates centrosome movement and that its heterozygous or homozygous deletion promotes aneuploidy and tumour formation. We found that Pten is recruited to pre-mitotic centrosomes in a Plk1-dependent fashion to create a docking site for protein complexes containing the PDZ-domain-containing protein Dlg1 (also known as Sap97) and Eg5 (also known as Kif11), a kinesin essential for centrosome movement and bipolar spindle formation4. Docking of Dlg1–Eg5 complexes to Pten depended on Eg5 phosphorylation by the Nek9–Nek6 mitotic kinase cascade and Cdk1. PDZ-BD deletion or Dlg1 ablation impaired loading of Eg5 onto centrosomes and spindle pole motility, yielding asymmetrical spindles that are prone to chromosome missegregation. Collectively, these data demonstrate that Pten, through the Dlg1-binding ability of its PDZ-BD, accumulates phosphorylated Eg5 at duplicated centrosomes to establish symmetrical bipolar spindles that properly segregate chromosomes, and suggest that this function contributes to tumour suppression. PMID:27240320

  18. From electron microscopy to molecular cell biology, molecular genetics and structural biology: intracellular transport and kinesin superfamily proteins, KIFs: genes, structure, dynamics and functions.

    PubMed

    Hirokawa, Nobutaka

    2011-01-01

    Cells transport and sort various proteins and lipids following synthesis as distinct types of membranous organelles and protein complexes to the correct destination at appropriate velocities. This intracellular transport is fundamental for cell morphogenesis, survival and functioning not only in highly polarized neurons but also in all types of cells in general. By developing quick-freeze electron microscopy (EM), new filamentous structures associated with cytoskeletons are uncovered. The characterization of chemical structures and functions of these new filamentous structures led us to discover kinesin superfamily molecular motors, KIFs. In this review, I discuss the identification of these new structures and characterization of their functions using molecular cell biology and molecular genetics. KIFs not only play significant roles by transporting various cargoes along microtubule rails, but also play unexpected fundamental roles on various important physiological processes such as learning and memory, brain wiring, development of central nervous system and peripheral nervous system, activity-dependent neuronal survival, development of early embryo, left-right determination of our body and tumourigenesis. Furthermore, by combining single-molecule biophysics with structural biology such as cryo-electrom microscopy and X-ray crystallography, atomic structures of KIF1A motor protein of almost all states during ATP hydrolysis have been determined and a common mechanism of motility has been proposed. Thus, this type of studies could be a good example of really integrative multidisciplinary life science in the twenty-first century.

  19. Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5*

    PubMed Central

    Liu, Liqiong; Richard, Jessica; Kim, Sunyoung; Wojcik, Edward J.

    2014-01-01

    Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable “next generation” target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are “druggable.” One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease. PMID:24737313

  20. A computational model predicts Xenopus meiotic spindle organization.

    PubMed

    Loughlin, Rose; Heald, Rebecca; Nédélec, François

    2010-12-27

    The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5-like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.

  1. The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants.

    PubMed

    Suetsugu, Noriyuki; Sato, Yoshikatsu; Tsuboi, Hidenori; Kasahara, Masahiro; Imaizumi, Takato; Kagawa, Takatoshi; Hiwatashi, Yuji; Hasebe, Mitsuyasu; Wada, Masamitsu

    2012-11-01

    Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.

  2. On the assembly of kinesin-based nanotransport systems

    NASA Astrophysics Data System (ADS)

    Oliveira, Daniel; Kim, Domyoung; Umetsu, Mitsuo; Adschiri, Tadafumi; Teizer, Winfried

    2013-03-01

    The ongoing pursuit to construct an artificial functional nanorobot has been preceded by biological equivalent long ago. Many proteins act at the nano-scale as biological motors for rotation or translation, being responsible for many fundamental processes. Among these proteins, kinesin is considered a promising tool in the development of synthetic nano-machines. The kinesin protein is a well known naturally occurring molecular machine capable of cargo transport upon interaction with cytoplasmic systems of fibers, known as microtubules. Conversion of chemical energy into mechanical work, harnessed by the hydrolysis of ATP, propels kinesin along microtubules. Even though recent efforts were made to engineer tailor-made artificial nanotransport systems using kinesin, no systematic study investigated how these systems can be built from the bottom up. Relying on the Surface Plasmon Resonance technique, we will show for the first time that it is possible to quantitatively evaluate how each component of such nanoscopic machines is sequentially assembled by monitoring the individual association of its components, specifically, the kinesin association to microtubule as well as the cargo-kinesin association.

  3. Monitoring spindle orientation: Spindle position checkpoint in charge.

    PubMed

    Caydasi, Ayse K; Ibrahim, Bashar; Pereira, Gislene

    2010-12-11

    Every cell division in budding yeast is inherently asymmetric and counts on the correct positioning of the mitotic spindle along the mother-daughter polarity axis for faithful chromosome segregation. A surveillance mechanism named the spindle position checkpoint (SPOC), monitors the orientation of the mitotic spindle and prevents cells from exiting mitosis when the spindle fails to align along the mother-daughter axis. SPOC is essential for maintenance of ploidy in budding yeast and similar mechanisms might exist in higher eukaryotes to ensure faithful asymmetric cell division. Here, we review the current model of SPOC activation and highlight the importance of protein localization and phosphorylation for SPOC function.

  4. Prometaphase spindle maintenance by an antagonistic motor-dependent force balance made robust by a disassembling lamin-B envelope

    PubMed Central

    Civelekoglu-Scholey, Gul; Tao, Li; Brust-Mascher, Ingrid; Wollman, Roy

    2010-01-01

    We tested the classical hypothesis that astral, prometaphase bipolar mitotic spindles are maintained by balanced outward and inward forces exerted on spindle poles by kinesin-5 and -14 using modeling of in vitro and in vivo data from Drosophila melanogaster embryos. Throughout prometaphase, puncta of both motors aligned on interpolar microtubules (MTs [ipMTs]), and motor perturbation changed spindle length, as predicted. Competitive motility of purified kinesin-5 and -14 was well described by a stochastic, opposing power stroke model incorporating motor kinetics and load-dependent detachment. Motor parameters from this model were applied to a new stochastic force-balance model for prometaphase spindles, providing a good fit to data from embryos. Maintenance of virtual spindles required dynamic ipMTs and a narrow range of kinesin-5 to kinesin-14 ratios matching that found in embryos. Functional perturbation and modeling suggest that this range can be extended significantly by a disassembling lamin-B envelope that surrounds the prometaphase spindle and augments the finely tuned, antagonistic kinesin force balance to maintain robust prometaphase spindles as MTs assemble and chromosomes are pushed to the equator. PMID:20065089

  5. Central Spindle Self-Organization and Cytokinesis in Artificially Activated Sea Urchin Eggs.

    PubMed

    Henson, John H; Buckley, Mary W; Yeterian, Mesrob; Weeks, Richard M; Simerly, Calvin R; Shuster, Charles B

    2016-04-01

    The ability of microtubules of the mitotic apparatus to control the positioning and initiation of the cleavage furrow during cytokinesis was first established from studies on early echinoderm embryos. However, the identity of the microtubule population that imparts cytokinetic signaling is unclear. The two main--and not necessarily mutually exclusive--candidates are the central spindle and the astral rays. In the present study, we examined cytokinesis in ammonia-activated sea urchin eggs, which lack paternally derived centrosomes and undergo mitosis mediated by unusual anastral, bipolar mini-spindles. Live cell imaging and immunolabeling for microtubules and the centralspindlin constituent and kinesin-related protein, MKLP1, demonstrated that furrowing in ammonia-activated eggs was associated with aligned arrays of centralspindlin-linked, opposed bundles of antiparallel microtubules. These autonomous, zipper-like arrays were not associated with a mitotic apparatus, but did possess characteristics similar to the central spindle region of control, fertilized embryos. Our results highlight the self-organizing nature of the central spindle region and its ability to induce cytokinesis-like furrowing, even in the absence of a complete mitotic apparatus. © 2016 Marine Biological Laboratory.

  6. Hice1, a Novel Microtubule-Associated Protein Required for Maintenance of Spindle Integrity and Chromosomal Stability in Human Cells▿ †

    PubMed Central

    Wu, Guikai; Lin, Yi-Tzu; Wei, Randy; Chen, Yumay; Shan, Zhiyin; Lee, Wen-Hwa

    2008-01-01

    Spindle integrity is critical for efficient mitotic progression and accurate chromosome segregation. Deregulation of spindles often leads to structural and functional aberrations, ultimately promoting segregation errors and aneuploidy, a hallmark of most human cancers. Here we report the characterization of a previously identified human sarcoma antigen (gene located at 19p13.11), Hice1, an evolutionarily nonconserved 46-kDa coiled-coil protein. Hice1 shows distinct cytoplasmic localization and associates with interphase centrosomes and mitotic spindles, preferentially at the spindle pole vicinity. Depletion of Hice1 by RNA interference resulted in abnormal and unstable spindle configurations, mitotic delay at prometaphase and metaphase, and elevated aneuploidy. Conversely, loss of Hice1 had minimal effects on interphase centrosome duplication. We also found that both full-length Hice1 and Hice1-N1, which is composed of 149 amino acids of the N-terminal region, but not the mutant lacking the N-terminal region, exhibited activities of microtubule bundling and stabilization at a near-physiological concentration. Consistently, overexpression of Hice1 rendered microtubule bundles in cells resistant to nocodazole- or cold-treatment-induced depolymerization. These results demonstrate that Hice1 is a novel microtubule-associated protein important for maintaining spindle integrity and chromosomal stability, in part by virtue of its ability to bind, bundle, and stabilize microtubules. PMID:18362163

  7. Bidirectional motility of the fission yeast kinesin-5, Cut7

    SciTech Connect

    Edamatsu, Masaki

    2014-03-28

    Highlights: • Motile properties of Cut7 (fission yeast kinesin-5) were studied for the first time. • Half-length Cut7 moved toward plus-end direction of microtubule. • Full-length Cut7 moved toward minus-end direction of microtubule. • N- and C-terminal microtubule binding sites did not switch the motile direction. - Abstract: Kinesin-5 is a homotetrameric motor with its motor domain at the N-terminus. Kinesin-5 crosslinks microtubules and functions in separating spindle poles during mitosis. In this study, the motile properties of Cut7, fission yeast kinesin-5, were examined for the first time. In in vitro motility assays, full-length Cut7 moved toward minus-end of microtubules, but the N-terminal half of Cut7 moved toward the opposite direction. Furthermore, additional truncated constructs lacking the N-terminal or C-terminal regions, but still contained the motor domain, did not switch the motile direction. These indicated that Cut7 was a bidirectional motor, and microtubule binding regions at the N-terminus and C-terminus were not involved in its directionality.

  8. Fbxo30 Regulates Mammopoiesis by Targeting the Bipolar Mitotic Kinesin Eg5

    PubMed Central

    Liu, Yan; Wang, Yin; Du, Zhanwen; Yan, Xiaoli; Zheng, Pan; Liu, Yang

    2016-01-01

    Fbxo30 is an orphan member of the F-box protein family with no known substrate or function. Here, we report that while Fbxo30−/− mice exhibit normal development, growth, life span, and fertility, the females fail to nurture their offspring due to defective mammopoiesis. Mass spectrometry analysis of Fbxo30-associated proteins revealed that Fbxo30 specifically interacts with the bipolar spindle kinesin EG5 (encoded by Kif11). As a result, Fbxo30 targets Eg5 for ubiquitinylation and controls its oscillation during the cell cycle. Correlated with EG5 dysregulation, Fbxo30−/− mammary epithelial cells exhibit multiple defects in centrosome homeostasis, mitotic spindle formation, and proliferation. Effects on proliferation, centrosome homeostasis, and mammopoiesis in the Fbxo30−/− mice were rescued through normalization of Eg5 activity using shRNA and/or an EG5 inhibitor. Our data reveal the Fbxo30-Eg5 interaction as a critical checkpoint in mammopoiesis and a critical role for ubiquitinylation-regulated Eg5 oscillation in the cell cycle. PMID:27117404

  9. Identification, molecular cloning, and transcription analysis of the Choristoneura fumiferana nuclear polyhedrosis virus spindle-like protein gene.

    PubMed

    Liu, J J; Carstens, E B

    1996-09-15

    The Choristoneura fumiferana nuclear polyhedrosis virus spindle-like protein (slp) gene has been identified and localized immediately downstream and in the same orientation as the CfMNPV DNA polymerase gene. The slp gene is 1101 bp long, predicted to code for a 366 amino acid (42.1 kDa) polypeptide. Transcriptional analysis revealed that the CfMNPV slp gene is expressed at late times postinfection, beginning at 24 hr postinfection and is most abundantly expressed after 36 hr. Transcription initiates within a single baculovirus consensus late start site sequence (GTAAG) at position -18 relative to the translation start codon. Based on amino acid comparisons, the CfMNPV gene is closely related to other similar baculovirus genes and distantly but recognizably related to the fusolin proteins of two entomopoxviruses. The conservation of amino acid sequence, glycosylation signals and specific domains throughout the protein suggest that this gene product may play an important role in insect DNA virus replication.

  10. Kinesin and Dynein Move a Peroxisome in Vivo: A Tug-of-War or Coordinated Movement?

    NASA Astrophysics Data System (ADS)

    Kural, Comert; Kim, Hwajin; Syed, Sheyum; Goshima, Gohta; Gelfand, Vladimir I.; Selvin, Paul R.

    2005-06-01

    We used fluorescence imaging with one nanometer accuracy (FIONA) to analyze organelle movement by conventional kinesin and cytoplasmic dynein in a cell. We located a green fluorescence protein (GFP)-tagged peroxisome in cultured Drosophila S2 cells to within 1.5 nanometers in 1.1 milliseconds, a 400-fold improvement in temporal resolution, sufficient to determine the average step size to be ~8 nanometers for both dynein and kinesin. Furthermore, we found that dynein and kinesin do not work against each other in vivo during peroxisome transport. Rather, multiple kinesins or multiple dyneins work together, producing up to 10 times the in vitro speed.

  11. Axonal spheroids in ovine neuroaxonal dystrophy are immunopositive to kinesin and dynein.

    PubMed

    Finnie, John W; Manavis, Jim

    2017-07-01

    Neuroaxonal dystrophy (NAD) is a neurologic disorder of sheep characterized by accumulation of numerous axonal swellings (spheroids) in specific regions of the brainstem and spinal cord. Disruption of axonal transport, which is driven in anterograde and retrograde directions by the molecular motors, kinesin and dynein, respectively, is believed to contribute to spheroid development. Accordingly, we examined spheroids in ovine NAD cases immunohistochemically for kinesin and dynein and found both motor proteins, with dynein more strongly expressed than kinesin. Further investigations of the kinesin and dynein content of axonal spheroids in NAD, and other neurodegenerative disorders of domestic animals, could assist in better understanding the pathogenesis of these diseases.

  12. MAPping out distribution routes for kinesin couriers.

    PubMed

    Atherton, Joseph; Houdusse, Anne; Moores, Carolyn

    2013-10-01

    In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long-distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarised, compartmentalised and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio-temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of sub-domain-specific microtubule (MT) tracks, sign-posted by different tubulin isoforms, tubulin post-translational modifications, tubulin GTPase activity and MT-associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with a particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that - especially for axonal cargo - alterations to the MT track can influence transport, although in vivo, it is likely that multiple track-based effects act synergistically to ensure accurate cargo distribution.

  13. Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

    PubMed

    Golsteyn, R M; Mundt, K E; Fry, A M; Nigg, E A

    1995-06-01

    Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

  14. Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1

    NASA Astrophysics Data System (ADS)

    Xu, Jing

    2013-03-01

    Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signalling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (approximately fourfold) without altering kinesin's single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results (Nat. Commun., 3:754, 2012) provide the first direct evidence of a protein kinase upregulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin. Work supported by NIGMS grants GM64624 to SPG, GM74830-06A1 to LH, GM76516 to LB, NS048501 to SJK, and AHA grant 825278F to JX.

  15. The ASQ2 gene required for mother-daughter centriole linkage and mitotic spindle orientation encodes a conserved TBCC-like protein

    PubMed Central

    Feldman, Jessica L.; Marshall, Wallace F.

    2009-01-01

    Summary An intriguing feature of centrioles is that these highly complicated microtubule-based structures duplicate once per cell cycle and the cell has precise control over their number. Each cell contains exactly two centrioles, linked together as a pair, one of which is a mother centriole formed in a previous cell cycle and the other a daughter centriole whose assembly is templated by the mother. Neither the molecular basis nor the functional role of mother-daughter centriole linkage is understood. We have identified a mutant, asq2, with defects in centriole linkage. asq2 mutant cells have variable numbers of centrioles and defects in centriole positioning. Here, we show that ASQ2 encodes the novel conserved protein, TBCCd1, a member of a protein family that includes a tubulin folding co-chaperone and the retinitis pigmentosa protein, RP2, involved in tubulin quality control during ciliogenesis. We characterize mitosis in asq2 cells. We show that the majority of cells establish a bipolar spindle, but that cells have defects in spindle orientation. A small subset of asq2 cells have centrioles at both poles, and these cells have properly positioned spindles, indicating that centrioles at the poles may be important for spindle orientation. The defects in centriole number control, centriole positioning, and spindle orientation appear to arise from a primary defect in centriole linkage mediated by TBCCd1/ASQ2. PMID:19631545

  16. PTEN regulates EG5 to control spindle architecture and chromosome congression during mitosis

    PubMed Central

    He, Jinxue; Zhang, Zhong; Ouyang, Meng; Yang, Fan; Hao, Hongbo; Lamb, Kristy L.; Yang, Jingyi; Yin, Yuxin; Shen, Wen H.

    2016-01-01

    Architectural integrity of the mitotic spindle is required for efficient chromosome congression and accurate chromosome segregation to ensure mitotic fidelity. Tumour suppressor PTEN has multiple functions in maintaining genome stability. Here we report an essential role of PTEN in mitosis through regulation of the mitotic kinesin motor EG5 for proper spindle architecture and chromosome congression. PTEN depletion results in chromosome misalignment in metaphase, often leading to catastrophic mitotic failure. In addition, metaphase cells lacking PTEN exhibit defects of spindle geometry, manifested prominently by shorter spindles. PTEN is associated and co-localized with EG5 during mitosis. PTEN deficiency induces aberrant EG5 phosphorylation and abrogates EG5 recruitment to the mitotic spindle apparatus, leading to spindle disorganization. These data demonstrate the functional interplay between PTEN and EG5 in controlling mitotic spindle structure and chromosome behaviour during mitosis. We propose that PTEN functions to equilibrate mitotic phosphorylation for proper spindle formation and faithful genomic transmission. PMID:27492783

  17. Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

    PubMed

    Margolis, R L; Rauch, C T; Pirollet, F; Job, D

    1990-12-01

    STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.

  18. Spindle microtubules generate tension-dependent changes in the distribution of inner kinetochore proteins

    PubMed Central

    Suzuki, Aussie; Hori, Tetsuya; Nishino, Tatsuya; Usukura, Jiro; Miyagi, Atsushi; Morikawa, Kosuke

    2011-01-01

    The kinetochore forms a dynamic interface with microtubules from the mitotic spindle. Live-cell light microscopy–based observations on the dynamic structural changes within the kinetochore suggest that molecular rearrangements within the kinetochore occur upon microtubule interaction. However, the source of these rearrangements is still unclear. In this paper, we analyze vertebrate kinetochore ultrastructure by immunoelectron microscopy (EM) in the presence or absence of tension from spindle microtubules. We found that the inner kinetochore region defined by CENP-A, CENP-C, CENP-R, and the C-terminal domain of CENP-T is deformed in the presence of tension, whereas the outer kinetochore region defined by Ndc80, Mis12, and CENP-E is not stretched even under tension. Importantly, based on EM, fluorescence microscopy, and in vitro analyses, we demonstrated that the N and C termini of CENP-T undergo a tension-dependent separation, suggesting that CENP-T elongation is at least partly responsible for changes in the shape of the inner kinetochore. PMID:21464230

  19. High-resolution tracking of microtubule motility driven by a single kinesin motor.

    PubMed Central

    Malik, F; Brillinger, D; Vale, R D

    1994-01-01

    Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions. Images PMID:8183952

  20. Lack of association between the Trp719Arg polymorphism in kinesin-like protein-6 and coronary artery disease in 19 case-control studies.

    PubMed

    Assimes, Themistocles L; Hólm, Hilma; Kathiresan, Sekar; Reilly, Muredach P; Thorleifsson, Gudmar; Voight, Benjamin F; Erdmann, Jeanette; Willenborg, Christina; Vaidya, Dhananjay; Xie, Changchun; Patterson, Chris C; Morgan, Thomas M; Burnett, Mary Susan; Li, Mingyao; Hlatky, Mark A; Knowles, Joshua W; Thompson, John R; Absher, Devin; Iribarren, Carlos; Go, Alan; Fortmann, Stephen P; Sidney, Stephen; Risch, Neil; Tang, Hua; Myers, Richard M; Berger, Klaus; Stoll, Monika; Shah, Svati H; Thorgeirsson, Gudmundur; Andersen, Karl; Havulinna, Aki S; Herrera, J Enrique; Faraday, Nauder; Kim, Yoonhee; Kral, Brian G; Mathias, Rasika A; Ruczinski, Ingo; Suktitipat, Bhoom; Wilson, Alexander F; Yanek, Lisa R; Becker, Lewis C; Linsel-Nitschke, Patrick; Lieb, Wolfgang; König, Inke R; Hengstenberg, Christian; Fischer, Marcus; Stark, Klaus; Reinhard, Wibke; Winogradow, Janina; Grassl, Martina; Grosshennig, Anika; Preuss, Michael; Schreiber, Stefan; Wichmann, H-Erich; Meisinger, Christa; Yee, Jean; Friedlander, Yechiel; Do, Ron; Meigs, James B; Williams, Gordon; Nathan, David M; MacRae, Calum A; Qu, Liming; Wilensky, Robert L; Matthai, William H; Qasim, Atif N; Hakonarson, Hakon; Pichard, Augusto D; Kent, Kenneth M; Satler, Lowell; Lindsay, Joseph M; Waksman, Ron; Knouff, Christopher W; Waterworth, Dawn M; Walker, Max C; Mooser, Vincent E; Marrugat, Jaume; Lucas, Gavin; Subirana, Isaac; Sala, Joan; Ramos, Rafael; Martinelli, Nicola; Olivieri, Oliviero; Trabetti, Elisabetta; Malerba, Giovanni; Pignatti, Pier Franco; Guiducci, Candace; Mirel, Daniel; Parkin, Melissa; Hirschhorn, Joel N; Asselta, Rosanna; Duga, Stefano; Musunuru, Kiran; Daly, Mark J; Purcell, Shaun; Eifert, Sandra; Braund, Peter S; Wright, Benjamin J; Balmforth, Anthony J; Ball, Stephen G; Ouwehand, Willem H; Deloukas, Panos; Scholz, Michael; Cambien, Francois; Huge, Andreas; Scheffold, Thomas; Salomaa, Veikko; Girelli, Domenico; Granger, Christopher B; Peltonen, Leena; McKeown, Pascal P; Altshuler, David; Melander, Olle; Devaney, Joseph M; Epstein, Stephen E; Rader, Daniel J; Elosua, Roberto; Engert, James C; Anand, Sonia S; Hall, Alistair S; Ziegler, Andreas; O'Donnell, Christopher J; Spertus, John A; Siscovick, David; Schwartz, Stephen M; Becker, Diane; Thorsteinsdottir, Unnur; Stefansson, Kari; Schunkert, Heribert; Samani, Nilesh J; Quertermous, Thomas

    2010-11-02

    We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455), and clinical coronary artery disease (CAD). Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with noncarriers. The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in 19 case-control studies of nonfatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports. A total of 17,000 cases and 39,369 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the 19 studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with noncarriers. Regression analyses and fixed-effects meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early-onset disease (younger than 50 years of age for men and younger than 60 years of age for women) compared with similarly aged controls as well as all non-European subgroups. The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study. Copyright © 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  1. Lack of association between the Trp719Arg polymorphism in kinesin-like protein 6 and coronary artery disease in 19 case-control studies

    PubMed Central

    Assimes, Themistocles L; Hólm, Hilma; Kathiresan, Sekar; Reilly, Muredach P; Thorleifsson, Gudmar; Voight, Benjamin F; Erdmann, Jeanette; Willenborg, Christina; Vaidya, Dhananjay; Xie, Changchun; Patterson, Chris C; Morgan, Thomas M; Burnett, Mary Susan; Li, Mingyao; Hlatky, Mark A; Knowles, Joshua W; Thompson, John R; Absher, Devin; Iribarren, Carlos; Go, Alan; Fortmann, Stephen P; Sidney, Stephen; Risch, Neil; Tang, Hua; Myers, Richard M; Berger, Klaus; Stoll, Monika; Shah, Svati H.; Thorgeirsson, Gudmundur; Andersen, Karl; Havulinna, Aki S; Herrera, J. Enrique; Faraday, Nauder; Kim, Yoonhee; Kral, Brian G.; Mathias, Rasika; Ruczinski, Ingo; Suktitipat, Bhoom; Wilson, Alexander F; Yanek, Lisa R.; Becker, Lewis C; Linsel-Nitschke, Patrick; Lieb, Wolfgang; König, Inke R; Hengstenberg, Christian; Fischer, Marcus; Stark, Klaus; Reinhard, Wibke; Winogradow, Janina; Grassl, Martina; Grosshennig, Anika; Preuss, Michael; Eifert, Sandra; Schreiber, Stefan; Wichmann, H-Erich; Meisinger, Christa; Yee, Jean; Friedlander, Yechiel; Do, Ron; Meigs, James B; Williams, Gordon; Nathan, David M; MacRae, Calum A; Qu, Liming; Wilensky, Robert L; Matthai, William H.; Qasim, Atif N; Hakonarson, Hakon; Pichard, Augusto D; Kent, Kenneth M; Satler, Lowell; Lindsay, Joseph M; Waksman, Ron; Knouff, Christopher W; Waterworth, Dawn M; Walker, Max C; Mooser, Vincent; Marrugat, Jaume; Lucas, Gavin; Subirana, Isaac; Sala, Joan; Ramos, Rafael; Martinelli, Nicola; Olivieri, Oliviero; Trabetti, Elisabetta; Malerba, Giovanni; Pignatti, Pier Franco; Guiducci, Candace; Mirel, Daniel; Parkin, Melissa; Hirschhorn, Joel N; Asselta, Rosanna; Duga, Stefano; Musunuru, Kiran; Daly, Mark J; Purcell, Shaun; Braund, Peter S; Wright, Benjamin J; Balmforth, Anthony J; Ball, Stephen G; Ouwehand, Willem H; Deloukas, Panos; Scholz, Michael; Cambien, Francois; Huge, Andreas; Scheffold, Thomas; Salomaa, Veikko; Girelli, Domenico; Granger, Christopher B.; Peltonen, Leena; McKeown, Pascal P; Altshuler, David; Melander, Olle; Devaney, Joseph M; Epstein, Stephen E; Rader, Daniel J; Elosua, Roberto; Engert, James C; Anand, Sonia S; Hall, Alistair S; Ziegler, Andreas; O’Donnell, Christopher J; Spertus, John A; Siscovick, David; Schwartz, Stephen M; Becker, Diane; Thorsteinsdottir, Unnur; Stefansson, Kari; Schunkert, Heribert; Samani, Nilesh J; Quertermous, Thomas

    2011-01-01

    Objectives We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455) and clinical coronary artery disease (CAD). Background Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with non-carriers. Methods The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in nineteen case-control studies of non-fatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports. Results Over 17 000 cases and 39 000 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the nineteen studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with non-carriers. Regression analyses and fixed effect meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early onset disease (<50 years of age for males and <60 years for females) compared with similarly aged controls as well as all non-European subgroups. Conclusions The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study. PMID:20933357

  2. Inhibition of the Motor Protein Eg5/Kinesin-5 in Amyloid β-Mediated Impairment of Hippocampal Long-Term Potentiation and Dendritic Spine Loss.

    PubMed

    Freund, Ronald K; Gibson, Emily S; Potter, Huntington; Dell'Acqua, Mark L

    2016-05-01

    Alzheimer's disease (AD) is characterized by neurofibrillary tangles, amyloid plaques, and neurodegeneration. However, this pathology is preceded by increased soluble amyloid beta (Aβ) 1-42 oligomers that interfere with the glutamatergic synaptic plasticity required for learning and memory, includingN-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). In particular, soluble Aβ(1-42) acutely inhibits LTP and chronically causes synapse loss. Many mechanisms have been proposed for Aβ-induced synaptic dysfunction, but we recently found that Aβ(1-42) inhibits the microtubule motor protein Eg5/kinesin-5. Here we compared the impacts of Aβ(1-42) and monastrol, a small-molecule Eg5 inhibitor, on LTP in hippocampal slices and synapse loss in neuronal cultures. Acute (20-minute) treatment with monastrol, like Aβ, completely inhibited LTP at doses >100 nM. In addition, 1 nM Aβ(1-42) or 50 nM monastrol inhibited LTP #x223c;50%, and when applied together caused complete LTP inhibition. At concentrations that impaired LTP, neither Aβ(1-42) nor monastrol inhibited NMDAR synaptic responses until #x223c;60 minutes, when only #x223c;25% inhibition was seen for monastrol, indicating that NMDAR inhibition was not responsible for LTP inhibition by either agent when applied for only 20 minutes. Finally, 48 hours of treatment with either 0.5-1.0μM Aβ(1-42) or 1-5μM monastrol reduced the dendritic spine/synapse density in hippocampal cultures up to a maximum of #x223c;40%, and when applied together at maximal concentrations, no additional spine loss resulted. Thus, monastrol can mimic and in some cases occlude the impact of Aβon LTP and synapse loss, suggesting that Aβinduces acute and chronic synaptic dysfunction in part through inhibiting Eg5. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  3. The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells.

    PubMed

    Chennathukuzhi, Vargheese; Morales, Carlos R; El-Alfy, Mohamed; Hecht, Norman B

    2003-12-23

    Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of the human protein Translin, is a widely expressed and highly conserved protein with proposed functions in chromosomal translocations, mitotic cell division, and mRNA transport, stabilization, and storage. Targeted inactivation of TB-RBP leads to abnormalities in fertility and behavior. A testis-enriched kinesin KIF17b coimmunoprecipitates with TB-RBP in a RNA-protein complex containing specific cAMP-responsive element modulator (CREM)-regulated mRNAs. The specificity of this interaction is confirmed by in vivo RNA-protein crosslinking and transfections of hippocampal neurons. Combining in situ hybridization and immunohistochemistry at the electron microscope level, a temporally sequential dissociation of KIF17b and TB-RBP from specific mRNAs is detected with TB-RBP release coincident with the time of mRNA translation, indicating a separation of the processes of transport and translation. We conclude that KIF17b serves as a molecular motor component of a TB-RBP-mouse ribonucleoprotein complex transporting a group of specific CREM-regulated mRNAs in mammalian male postmeiotic germ cells. Because KIF17b has been reported to control CREM-dependent transcription in male germ cells by regulating the intracellular location of the transcriptional coactivator activator of CREM in testis, this indicates that one kinesin links the processes of transcription and transport of specific mRNAs in mammalian male germ cells.

  4. Common general anesthetic propofol impairs kinesin processivity.

    PubMed

    Bensel, Brandon M; Guzik-Lendrum, Stephanie; Masucci, Erin M; Woll, Kellie A; Eckenhoff, Roderic G; Gilbert, Susan P

    2017-05-23

    Propofol is the most widely used i.v. general anesthetic to induce and maintain anesthesia. It is now recognized that this small molecule influences ligand-gated channels, including the GABAA receptor and others. Specific propofol binding sites have been mapped using photoaffinity ligands and mutagenesis; however, their precise target interaction profiles fail to provide complete mechanistic underpinnings for the anesthetic state. These results suggest that propofol and other common anesthetics, such as etomidate and ketamine, may target additional protein networks of the CNS to contribute to the desired and undesired anesthesia end points. Some evidence for anesthetic interactions with the cytoskeleton exists, but the molecular motors have received no attention as anesthetic targets. We have recently discovered that propofol inhibits conventional kinesin-1 KIF5B and kinesin-2 KIF3AB and KIF3AC, causing a significant reduction in the distances that these processive kinesins can travel. These microtubule-based motors are highly expressed in the CNS and the major anterograde transporters of cargos, such as mitochondria, synaptic vesicle precursors, neurotransmitter receptors, cell signaling and adhesion molecules, and ciliary intraflagellar transport particles. The single-molecule results presented show that the kinesin processive stepping distance decreases 40-60% with EC50 values <100 nM propofol without an effect on velocity. The lack of a velocity effect suggests that propofol is not binding at the ATP site or allosteric sites that modulate microtubule-activated ATP turnover. Rather, we propose that a transient propofol allosteric site forms when the motor head binds to the microtubule during stepping.

  5. Tubulin protofilaments and kinesin-dependent motility

    PubMed Central

    1992-01-01

    Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition

  6. Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase.

    PubMed

    Goldstein, Alina; Siegler, Nurit; Goldman, Darya; Judah, Haim; Valk, Ervin; Kõivomägi, Mardo; Loog, Mart; Gheber, Larisa

    2017-09-01

    The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.

  7. A tumor suppressor role of the Bub3 spindle checkpoint protein after apoptosis inhibition

    PubMed Central

    Moutinho-Santos, Tatiana

    2013-01-01

    Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor. PMID:23609535

  8. Potential involvement of kinesin-1 in the regulation of subcellular localization of Girdin

    SciTech Connect

    Muramatsu, Aya; Enomoto, Atsushi; Kato, Takuya; Weng, Liang; Kuroda, Keisuke; Asai, Naoya; Asai, Masato; Mii, Shinji; Takahashi, Masahide

    2015-08-07

    Girdin is an actin-binding protein that has multiple functions in postnatal neural development and cancer progression. We previously showed that Girdin is a regulator of migration for neuroblasts born from neural stem cells in the subventricular zone (SVZ) and the dentate gyrus of the hippocampus in the postnatal brain. Despite a growing list of Girdin-interacting proteins, the mechanism of Girdin-mediated migration has not been fully elucidated. Girdin interacts with Disrupted-In-Schizophrenia 1 and partitioning-defective 3, both of which have been shown to interact with the kinesin microtubule motor proteins. Based on this, we have identified that Girdin also interacts with kinesin-1, a member of neuronal kinesin proteins. Although a direct interaction of Girdin and kinesin-1 has not been determined, it is of interest to find that Girdin loss-of-function mutant mice with the mutation of a basic amino acid residue-rich region (Basic mut mice) exhibit limited interaction with kinesin-1. Furthermore, expression of a kinesin-1 mutant with motor defects, leads to Girdin mislocalization. Finally, consistent with previous studies on the role of kinesin proteins in trafficking a cell–cell adhesion molecule N-cadherin, Basic mut mice showed an aberrant expression pattern of N-cadherin in migrating SVZ neuroblasts. These findings suggest a potential role of Girdin/kinesin-1 interaction in the regulation of neuroblast migration in the postnatal brain. - Highlights: • Girdin is a regulator of migration for neuroblasts in the postnatal brain. • Girdin interacts with kinesin-1, a member of neuronal kinesin proteins. • Girdin mutant mice showed an aberrant expression of N-cadherin in neuroblasts.

  9. SAP-like domain in nucleolar spindle associated protein mediates mitotic chromosome loading as well as interphase chromatin interaction

    SciTech Connect

    Verbakel, Werner; Carmeliet, Geert; Engelborghs, Yves

    2011-08-12

    Highlights: {yields} The SAP-like domain in NuSAP is a functional DNA-binding domain with preference for dsDNA. {yields} This SAP-like domain is essential for chromosome loading during early mitosis. {yields} NuSAP is highly dynamic on mitotic chromatin, as evident from photobleaching experiments. {yields} The SAP-like domain also mediates NuSAP-chromatin interaction in interphase nucleoplasm. -- Abstract: Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy. The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase Nu

  10. Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells.

    PubMed

    Wilson, Meredith H; Holzbaur, Erika L F

    2015-01-01

    During skeletal muscle development, nuclei move dynamically through myotubes in a microtubule-dependent manner, driven by the microtubule motor protein kinesin-1. Loss of kinesin-1 leads to improperly positioned nuclei in culture and in vivo. Two models have been proposed to explain how kinesin-1 functions to move nuclei in myotubes. In the cargo model, kinesin-1 acts directly from the surface of the nucleus, whereas in an alternative model, kinesin-1 moves nuclei indirectly by sliding anti-parallel microtubules. Here, we test the hypothesis that an ensemble of Kif5B motors acts from the nuclear envelope to distribute nuclei throughout the length of syncytial myotubes. First, using an inducible dimerization system, we show that controlled recruitment of truncated, constitutively active kinesin-1 motors to the nuclear envelope is sufficient to prevent the nuclear aggregation resulting from depletion of endogenous kinesin-1. Second, we identify a conserved kinesin light chain (KLC)-binding motif in the nuclear envelope proteins nesprin-1 and nesprin-2, and show that recruitment of the motor complex to the nucleus via this LEWD motif is essential for nuclear distribution. Together, our findings demonstrate that the nucleus is a kinesin-1 cargo in myotubes and that nesprins function as nuclear cargo adaptors. The importance of achieving and maintaining proper nuclear position is not restricted to muscle fibers, suggesting that the nesprin-dependent recruitment of kinesin-1 to the nuclear envelope through the interaction of a conserved LEWD motif with kinesin light chain might be a general mechanism for cell-type-specific nuclear positioning during development.

  11. Kinesin Kip2 enhances microtubule growth in vitro through length-dependent feedback on polymerization and catastrophe

    PubMed Central

    Hibbel, Anneke; Bogdanova, Aliona; Mahamdeh, Mohammed; Jannasch, Anita; Storch, Marko; Schäffer, Erik; Liakopoulos, Dimitris; Howard, Jonathon

    2015-01-01

    The size and position of mitotic spindles is determined by the lengths of their constituent microtubules. Regulation of microtubule length requires feedback to set the balance between growth and shrinkage. Whereas negative feedback mechanisms for microtubule length control, based on depolymerizing kinesins and severing proteins, have been studied extensively, positive feedback mechanisms are not known. Here, we report that the budding yeast kinesin Kip2 is a microtubule polymerase and catastrophe inhibitor in vitro that uses its processive motor activity as part of a feedback loop to further promote microtubule growth. Positive feedback arises because longer microtubules bind more motors, which walk to the ends where they reinforce growth and inhibit catastrophe. We propose that positive feedback, common in biochemical pathways to switch between signaling states, can also be used in a mechanical signaling pathway to switch between structural states, in this case between short and long polymers. DOI: http://dx.doi.org/10.7554/eLife.10542.001 PMID:26576948

  12. Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1

    PubMed Central

    Xu, Jing; Reddy, Babu J. N.; Anand, Preetha; Shu, Zhanyong; Cermelli, Silvia; Mattson, Michelle K.; Tripathy, Suvranta K.; Hoss, Matthew T.; James, Nikita S.; King, Stephen J.; Huang, Lan; Bardwell, Lee; Gross, Steven P.

    2013-01-01

    Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results provide the first direct evidence of a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin. PMID:22453827

  13. Distinct Interaction Modes of the Kinesin-13 Motor Domain with the Microtubule

    PubMed Central

    Chatterjee, Chandrima; Benoit, Matthieu P.M.H.; DePaoli, Vania; Diaz-Valencia, Juan D.; Asenjo, Ana B.; Gerfen, Gary J.; Sharp, David J.; Sosa, Hernando

    2016-01-01

    Kinesins-13s are members of the kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other kinesins, kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile kinesins. PMID:27074684

  14. Biochemical perturbations of the mitotic spindle in Xenopus extracts using a diffusion-based microfluidic assay

    PubMed Central

    Yoo, Byung-Kuk; Buguin, Axel; Gueroui, Zoher

    2015-01-01

    A microfluidic device is a powerful tool to manipulate in a controlled manner at spatiotemporal scales for biological systems. Here, we describe a simple diffusion-based assay to generate and measure the effect of biochemical perturbations within the cytoplasm of cell-free extracts from Xenopus eggs. Our approach comprises a microliter reservoir and a model cytoplasm that are separated by a synthetic membrane containing sub-micrometric pores through which small molecules and recombinant proteins can diffuse. We have used this system to examine the perturbation of elements of the mitotic spindle, which is a microtubule-based bipolar structure involved in the segregation of the replicated genome to daughter cells during cell division. First, we used the small molecule inhibitor monastrol to target kinesin-5, a molecular motor that maintains the microtubule spindle bipolarity. Next, we explored the dynamics of the mitotic spindle by monitoring the exchange between unpolymerized and polymerized tubulin within microtubule fibers. These results show that a simple diffusion-based system can generate biochemical perturbations directly within a cell-free cytoplasm based on Xenopus egg extracts at the time scale of minutes. Our assay is therefore suitable for monitoring the dynamics of supramolecular assemblies within cell-free extracts in response to perturbations. This strategy opens up broad perspectives including phenotype screening or mechanistic studies of biological assembly processes and could be applied to other cell-free extracts such as those derived from mammalian or bacterial cells. PMID:26221196

  15. Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility.

    PubMed

    Yamagishi, Masahiko; Shigematsu, Hideki; Yokoyama, Takeshi; Kikkawa, Masahide; Sugawa, Mitsuhiro; Aoki, Mari; Shirouzu, Mikako; Yajima, Junichiro; Nitta, Ryo

    2016-08-02

    Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Ras transformation uncouples the kinesin-coordinated cellular nutrient response

    PubMed Central

    Zaganjor, Elma; Weil, Lauren M.; Gonzales, Joshua X.; Minna, John D.; Cobb, Melanie H.

    2014-01-01

    The kinesin family members (KIFs) KIF2A and KIF2C depolymerize microtubules, unlike the majority of other kinesins, which transport cargo along microtubules. KIF2A regulates the localization of lysosomes in the cytoplasm, which assists in activation of the mechanistic target of rapamycin complex 1 (mTORC1) on the lysosomal surface. We find that the closely related kinesin KIF2C also influences lysosomal organization in immortalized human bronchial epithelial cells (HBECs). Expression of KIF2C and, to a lesser extent, KIF2A in untransformed and mutant K-Ras–transformed cells is regulated by ERK1/2. Prolonged inhibition of ERK1/2 activation with PD0325901 mimics nutrient deprivation by disrupting lysosome organization and decreasing mTORC1 activity in HBEC, suggesting a long-term mechanism for optimization of mTORC1 activity by ERK1/2. We tested the hypothesis that up-regulation of KIF2C and KIF2A by ERK1/2 caused aberrant lysosomal positioning and mTORC1 activity in a mutant K-Ras–dependent cancer and cancer model. In Ras-transformed cells, however, mTORC1 activity and lysosome organization appear independent of ERK1/2 and these kinesins although ERK1/2 activity and the kinesins are required for Ras-dependent proliferation and migration. We conclude that mutant K-Ras repurposes these signaling and regulatory proteins to support the transformed phenotype. PMID:25002494

  17. Identification and characterization of INMAP, a novel interphase nucleus and mitotic apparatus protein that is involved in spindle formation and cell cycle progression

    SciTech Connect

    Shen, Enzhi; Lei, Yan; Liu, Qian; Zheng, Yanbo; Song, Chunqing; Marc, Jan; Wang, Yongchao; Sun, Le; Liang, Qianjin

    2009-04-15

    A novel protein that associates with interphase nucleus and mitotic apparatus (INMAP) was identified by screening HeLa cDNA expression library with an autoimmune serum followed by tandem mass spectrometry. Its complete cDNA sequence of 1.818 kb encodes 343 amino acids with predicted molecular mass of 38.2 kDa and numerous phosphorylation sites. The sequence is identical with nucleotides 1-1800 bp of an unnamed gene (GenBank accession no. (7022388)) and highly homologous with the 3'-terminal sequence of POLR3B. A monoclonal antibody against INMAP reacted with similar proteins in S. cerevisiae, Mel and HeLa cells, suggesting that it is a conserved protein. Confocal microscopy using either GFP-INMAP fusion protein or labeling with the monoclonal antibody revealed that the protein localizes as distinct dots in the interphase nucleus, but during mitosis associates closely with the spindle. Double immunolabeling using specific antibodies showed that the INMAP co-localizes with {alpha}-tubulin, {gamma}-tubulin, and NuMA. INMAP also co-immunoprecipitated with these proteins in their native state. Stable overexpression of INMAP in HeLa cell lines leads to defects in the spindle, mitotic arrest, formation of polycentrosomal and multinuclear cells, inhibition of growth, and apoptosis. We propose that INMAP is a novel protein that plays essential role in spindle formation and cell-cycle progression.

  18. Identification of MAC1: A Small Molecule That Rescues Spindle Bipolarity in Monastrol-Treated Cells.

    PubMed

    Al-Obaidi, Naowras; Mitchison, Timothy J; Crews, Craig M; Mayer, Thomas U

    2016-06-17

    The genetic integrity of each organism is intimately tied to the correct segregation of its genome during mitosis. Insights into the underlying mechanisms are fundamental for both basic research and the development of novel strategies to treat mitosis-relevant diseases such as cancer. Due to their fast mode of action, small molecules are invaluable tools to dissect mitosis. Yet, there is a great demand for novel antimitotic compounds. We performed a chemical genetic suppression screen to identify compounds that restore spindle bipolarity in cells treated with Monastrol, an inhibitor of the mitotic kinesin Eg5. We identified one compound-MAC1-that rescued spindle bipolarity in cells lacking Eg5 activity. Mechanistically, MAC1 induces the formation of additional microtubule nucleation centers, which allows kinesin Kif15-dependent bipolar spindle assembly in the absence of Eg5 activity. Thus, our chemical genetic suppression screen revealed novel unexpected insights into the mechanism of spindle assembly in mammalian cells.

  19. Dnt1 acts as a mitotic inhibitor of the spindle checkpoint protein dma1 in fission yeast

    PubMed Central

    Wang, Yamei; Li, Wen-zhu; Johnson, Alyssa E.; Luo, Zhou-qing; Sun, Xue-li; Feoktistova, Anna; McDonald, W. Hayes; McLeod, Ian; Yates, John R.; Gould, Kathleen L.; McCollum, Dannel; Jin, Quan-wen

    2012-01-01

    The Schizosaccharomyces pombe checkpoint protein Dma1 couples mitotic progression with cytokinesis and is important in delaying mitotic exit and cytokinesis when kinetochores are not properly attached to the mitotic spindle. Dma1 is a ubiquitin ligase and potential functional relative of the human tumor suppressor Chfr. Dma1 delays mitotic exit and cytokinesis by ubiquitinating a scaffold protein (Sid4) of the septation initiation network, which, in turn, antagonizes the ability of the Polo-like kinase Plo1 to promote cell division. Here we identify Dnt1 as a Dma1-binding protein. Several lines of evidence indicate that Dnt1 inhibits Dma1 function during metaphase. First, Dnt1 interacts preferentially with Dma1 during metaphase. Second, Dma1 ubiquitin ligase activity and Sid4 ubiquitination are elevated in dnt1∆ cells. Third, the enhanced mitotic defects in dnt1Δ plo1 double mutants are partially rescued by deletion of dma1+, suggesting that the defects in dnt1∆ plo1 double mutants are attributable to excess Dma1 activity. Taken together, these data show that Dnt1 acts to restrain Dma1 activity in early mitosis to allow normal mitotic progression. PMID:22809626

  20. The role of Hklp2 in the stabilization and maintenance of spindle bipolarity.

    PubMed

    Vanneste, David; Takagi, Masatoshi; Imamoto, Naoko; Vernos, Isabelle

    2009-11-03

    Spindle bipolarity relies on a fine balance of forces exerted by various molecular motors [1-4]. In most animal cells, spindle bipolarity requires sustained outward forces to push the spindle poles apart, an activity that is provided by Eg5, a conserved homotetrameric plus-end-directed kinesin that crosslinks and slides antiparallel microtubules apart [5]. These pushing forces are balanced by inward minus-end-directed forces. Impairing both Eg5 and dynein restores the formation of functional bipolar spindles [4], although the mechanism at play is far from clear. The current model also fails to explain why in some systems Eg5 inhibition does not promote bipolar spindle collapse [6, 7] or why increasing Eg5 levels does not interfere with bipolar spindle assembly [8]. Moreover, the C. elegans Eg5 ortholog is not required for bipolar spindle formation [9]. We show here that the kinesin Hklp2 participates in the assembly and stabilization of the bipolar spindle. Hklp2 localizes to the mitotic microtubules in a TPX2-dependent manner and to the chromosomes through Ki67. Our data indicate that its mechanism of action is clearly distinct from and complementary to that of Eg5, providing an additional understanding of the mechanism driving the formation and maintenance of the bipolar spindle.

  1. Kinesin-2: a family of heterotrimeric and homodimeric motors with diverse intracellular transport functions.

    PubMed

    Scholey, Jonathan M

    2013-01-01

    Kinesin-2 was first purified as a heterotrimeric, anterograde, microtubule-based motor consisting of two distinct kinesin-related subunits and a novel associated protein (KAP) that is currently best known for its role in intraflagellar transport and ciliogenesis. Subsequent work, however, has revealed diversity in the oligomeric state of different kinesin-2 motors owing to the combinatorial heterodimerization of its subunits and the coexistence of both heterotrimeric and homodimeric kinesin-2 motors in some cells. Although the functional significance of the homo- versus heteromeric organization of kinesin-2 motor subunits and the role of KAP remain uncertain, functional studies suggest that cooperation between different types of kinesin-2 motors or between kinesin-2 and a member of a different motor family can generate diverse patterns of anterograde intracellular transport. Moreover, despite being restricted to ciliated eukaryotes, kinesin-2 motors are now known to drive diverse transport events outside cilia. Here, I review the organization, assembly, phylogeny, biological functions, and motility mechanism of this diverse family of intracellular transport motors.

  2. Casein Kinase 2 Reverses Tail-Independent Inhibition of Kinesin-1

    NASA Astrophysics Data System (ADS)

    Xu, Jing; Shu, Zhanyong; Anand, Preetha; Reddy, Babu; Cermelli, Silvia; Whisenant, Thomas; King, Stephen; Bardwell, Lee; Huang, Lan; Gross, Steven

    2011-03-01

    Kinesin-1 is a plus-end microtubule-based molecular motor, and defects in kinesin transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail interaction, but is believed to be active otherwise. In contrast, this study uncovers a fast but reversible inhibition distinct from the canonical auto-inhibition pathway. The majority of the initially active kinesin (full-length or tail-less) loses its ability to bind/interact with microtubule, and Casein Kinase 2 (CK2) reverses this inactivation (up to 4-fold) without altering kinesin's single motor properties. Motor phosphorylation is not required for this CK2 -mediated kinesin activation. In cultured mammalian cells, knockdown of CK2 level, but not kinase activity, was sufficient to decrease the force required to stall lipid droplet transport, consistent with a reduction in the number of active motors. We propose that CK2 forms a positive regulating complex with the motor. This study provides the first direct evidence of a protein kinase positively regulating kinesin-transport, and uncovers a pathway whereby inactive cargo-bound kinesin can be activated. This work is supported by NIGMS grants GM64624 and GM079156 to SPG, GM-74830 to LH, NIH grants GM76516 and GM60366 to LB, and AHA grant 825278F to JX.

  3. Localization of the Microtubule End Binding Protein EB1 Reveals Alternative Pathways of Spindle Development in Arabidopsis Suspension CellsW⃞

    PubMed Central

    Chan, Jordi; Calder, Grant; Fox, Samantha; Lloyd, Clive

    2005-01-01

    In a previous study on Arabidopsis thaliana suspension cells transiently infected with the microtubule end binding protein AtEB1a–green fluorescent protein (GFP), we reported that interphase microtubules grow from multiple sites dispersed over the cortex, with plus ends forming the characteristic comet-like pattern. In this study, AtEB1a-GFP was used to study the transitions of microtubule arrays throughout the division cycle of cells lacking a defined centrosome. During division, the dispersed origin of microtubules was replaced by a more focused pattern with the plus end comets growing away from sites associated with the nuclear periphery. The mitotic spindle then evolved in two quite distinct ways depending on the presence or absence of the preprophase band (PPB): the cells displaying outside-in as well as inside-out mitotic pathways. In those cells possessing a PPB, the fusion protein labeled material at the nuclear periphery that segregated into two polar caps, perpendicular to the PPB, before nuclear envelope breakdown (NEBD). These polar caps then marked the spindle poles upon NEBD. However, in the population of cells without PPBs, there was no prepolarization of material at the nuclear envelope before NEBD, and the bipolar spindle only emerged clearly after NEBD. Such cells had variable spindle orientations and enhanced phragmoplast mobility, suggesting that the PPB is involved in a polarization event that promotes early spindle pole morphogenesis and subsequent positional stability during division. Astral-like microtubules are not usually prominent in plant cells, but they are clearly seen in these Arabidopsis cells, and we hypothesize that they may be involved in orienting the division plane, particularly where the plane is not determined before division. PMID:15879559

  4. Detection and quantification of microtubule detachment from centrosomes and spindle poles.

    PubMed

    Ganguly, Anutosh; Yang, Hailing; Cabral, Fernando

    2013-01-01

    Microtubule detachment from microtubule organizing centers is an important cellular process required for normal cell proliferation. When cells enter mitosis, microtubule turnover increases along with a concurrent increase in microtubule detachment. MCAK, a kinesin-related protein whose abundance is highest during the early stages of mitosis, has been shown to regulate microtubule detachment. Abnormal increases or decreases in the frequency of detachment interfere with spindle function and inhibit cell division. It has been shown that drugs able to promote microtubule assembly (e.g., paclitaxel, epothilones) prevent cell division by suppressing microtubule detachment from centrosomes. Conversely, cytotoxic concentrations of microtubule destabilizing drugs (e.g., vinblastine, nocodazole), tubulin mutations that cause paclitaxel resistance, and specific β-tubulin isotypes increase the frequency of microtubule detachment. In this chapter, we describe a method to calculate the frequency of microtubule detachment by transfecting cells with EGFP-MAP4 and directly observing detachment by live cell imaging.

  5. Patterns of kinesin evolution reveal a complex ancestral eukaryote with a multifunctional cytoskeleton

    PubMed Central

    2010-01-01

    Background The genesis of the eukaryotes was a pivotal event in evolution and was accompanied by the acquisition of numerous new cellular features including compartmentalization by cytoplasmic organelles, mitosis and meiosis, and ciliary motility. Essential for the development of these features was the tubulin cytoskeleton and associated motors. It is therefore possible to map ancient cell evolution by reconstructing the evolutionary history of motor proteins. Here, we have used the kinesin motor repertoire of 45 extant eukaryotes to infer the ancestral state of this superfamily in the last common eukaryotic ancestor (LCEA). Results We bioinformatically identified 1624 putative kinesin proteins, determined their protein domain architectures and calculated a comprehensive Bayesian phylogeny for the kinesin superfamily with statistical support. These data enabled us to define 51 anciently-derived kinesin paralogs (including three new kinesin families) and 105 domain architectures. We then mapped these characters across eukaryotes, accounting for secondary loss within established eukaryotic groupings, and alternative tree topologies. Conclusions We show that a minimum of 11 kinesin families and 3 protein domain architectures were present in the LCEA. This demonstrates that the microtubule-based cytoskeleton of the LCEA was surprisingly highly developed in terms of kinesin motor types, but that domain architectures have been extensively modified during the diversification of the eukaryotes. Our analysis provides molecular evidence for the existence of several key cellular functions in the LCEA, and shows that a large proportion of motor family diversity and cellular complexity had already arisen in this ancient cell. PMID:20423470

  6. Dermal plexiform spindle cell lipoma.

    PubMed

    Val-Bernal, José Fernando; Hermana, Sandra

    2016-01-01

    Spindle cell lipoma located in the dermis is uncommon. The plexiform variant of this tumor is rare. In fact, only six cases of this variant have been described previously. We report herein a case of dermal plexiform spindle cell lipoma with prominent myxoid matrix. A 47-year-old male patient presented with a solitary, 2.2 cm-cutaneous mass in the right buttock region that had slowly increased in size for over one year. The dermal lesion was characterized by a mixture of mature adipocytes, spindle shaped cells and inconspicuous ropey collagen bundles in a mucinous background. This lesion showed a fascicular and plexiform pattern with adipocytes irregularly arranged, predominant in the depth of the lesion. Immunohistochemically, the spindle cells were positive for CD34, factor XIIIa, and vimentin, and negative for retinoblastoma protein, claudin-1, GLUT-1, epithelial membrane antigen, neurofilament protein, and Sox-10. S100 protein stained a thinned cytoplasmic rim of mature adipocytes and labeled about 25% of spindle cells in the most superficial areas. A review of the seven cases published, including the present report, revealed that there were five females and two males. Most cases located in the thigh-groin-buttock area. The age of the patients ranged from 32 to 58 years with a mean of 45.7 years. Clinical diagnosis suggested a lipomatous or neural tumor in six cases. The main differential diagnosis includes dermal intraneural plexiform neurofibroma and purely intradermal monophasic plexiform spindle cell nevus.

  7. Spindle checkpoint protein Bub1 corrects mitotic aberrancy induced by human T-cell leukemia virus type I Tax.

    PubMed

    Sasaki, M; Sugimoto, K; Tamayose, K; Ando, M; Tanaka, Y; Oshimi, K

    2006-06-22

    Bub1 is a component of the mitotic spindle checkpoint apparatus. Abnormality of this apparatus is known to cause multinuclei formation, a hallmark of chromosomal instability (CIN). A549, aneuploid cell line, aberrantly passed through the mitotic phase and became multinuclei morphology in the presence of nocodazole. Time-lapse videomicroscopy showed unreported bizarre morphology, which we named 'mitotic lobulation' in A549 cells just before the exit from mitosis and multinuclei formation. External expression of wild-type Bub1-EGFP clearly suppressed the multinuclei formation by retaining A549 cells at the mitotic phase during 48 h of time-lapse observation. This suppressive effect on mitotic aberrancy should not be mere restoration of normal Bub1 function, because A549 cells express proper amount of Bub1, which distributed cytoplasm during interphase and concentrated at kinetochore in metaphase. Furthermore, external expression of wild-type Bub1-EGFP suppressed multinuclei formation induced by Tax both in A549 and HeLa cells. Tax is known to induce mitotic abnormality by binding and inactivating Mad1. These observations, therefore, suggest functional redundancy between Bub1 and other mitotic checkpoint protein(s) and a possibility of correction of mitotic aberrancy by external Bub1 expression.

  8. The Spindle Pole Body Protein Cdc11p Links Sid4p to the Fission Yeast Septation Initiation Network

    PubMed Central

    Tomlin, Gregory C.; Morrell, Jennifer L.; Gould, Kathleen L.

    2002-01-01

    The Schizosaccharomyces pombe septation initiation network (SIN) signals the onset of cell division from the spindle pole body (SPB) and is regulated by the small GTPase Spg1p. The localization of SIN components including Spg1p to the SPB is required for cytokinesis and is dependent on Sid4p, a constitutive resident of SPBs. However, a direct interaction between Sid4p and other members of the SIN has not been detected. To understand how Sid4p is linked to other SIN components, we have begun to characterize an S. pombe homolog of the Saccharomyces cerevisiae SPB protein Nud1p. We have determined that this S. pombe Nud1p homolog corresponds to Cdc11p, a previously uncharacterized SIN element. We report that Cdc11p is present constitutively at SPBs and that its function appears to be required for the localization of all other SIN components to SPBs with the exception of Sid4p. The Cdc11p C terminus localizes the protein to SPBs in a Sid4p-dependent manner, and we demonstrate a direct Cdc11p-Sid4p interaction. The N-terminus of Cdc11p is required for Spg1p binding to SPBs. Our studies indicate that Cdc11p provides a physical link between Sid4p and the Spg1p signaling pathway. PMID:11950932

  9. The SUN Protein Mps3 Is Required for Spindle Pole Body Insertion into the Nuclear Membrane and Nuclear Envelope Homeostasis

    PubMed Central

    Smoyer, Christine J.; McCroskey, Scott; Miller, Brandon D.; Weaver, Kyle J.; Delventhal, Kym M.; Unruh, Jay; Slaughter, Brian D.; Jaspersen, Sue L.

    2011-01-01

    The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition. PMID:22125491

  10. Centrin: Another target of monastrol, an inhibitor of mitotic spindle

    NASA Astrophysics Data System (ADS)

    Duan, Lian; Wang, Tong-Qing; Bian, Wei; Liu, Wen; Sun, Yue; Yang, Bin-Sheng

    2015-02-01

    Monastrol, a cell-permeable inhibitor, considered to specifically inhibit kinesin Eg5, can cause mitotic arrest and monopolar spindle formation, thus exhibiting antitumor properties. Centrin, a ubiquitous protein associated with centrosome, plays a critical role in centrosome duplication. Moreover, a correlation between centrosome amplification and cancer has been reported. In this study, it is proposed for the first time that centrin may be another target of the anticancer drug monastrol since monastrol can effectively inhibit not only the growth of the transformed Escherichia coli cells in vivo, but also the Lu3+-dependent self-assembly of EoCen in vitro. The two closely related compounds (Compounds 1 and 2) could not take the same effect. Fluorescence titration experiments suggest that four monastrols per protein is the optimum binding pattern, and the binding constants at different temperatures were obtained. Detailed thermodynamic analysis indicates that hydrophobic force is the main acting force between monastrol and centrin, and the extent of monastrol inhibition of centrin self-assembly is highly dependent upon the hydrophobic region of the protein, which is largely exposed by the binding of metal ions.

  11. Budding yeast dma proteins control septin dynamics and the spindle position checkpoint by promoting the recruitment of the Elm1 kinase to the bud neck.

    PubMed

    Merlini, Laura; Fraschini, Roberta; Boettcher, Barbara; Barral, Yves; Lucchini, Giovanna; Piatti, Simonetta

    2012-01-01

    The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC). This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.

  12. Genome-wide RNAi screen for synthetic lethal interactions with the C. elegans kinesin-5 homolog BMK-1

    PubMed Central

    Maia, André F.; Tanenbaum, Marvin E.; Galli, Matilde; Lelieveld, Daphne; Egan, David A.; Gassmann, Reto; Sunkel, Claudio E.; van den Heuvel, Sander; Medema, René H.

    2015-01-01

    Kinesins are a superfamily of microtubule-based molecular motors that perform various transport needs and have essential roles in cell division. Among these, the kinesin-5 family has been shown to play a major role in the formation and maintenance of the bipolar mitotic spindle. Moreover, recent work suggests that kinesin-5 motors may have additional roles. In contrast to most model organisms, the sole kinesin-5 gene in Caenorhabditis elegans, bmk-1, is not required for successful mitosis and animals lacking bmk-1 are viable and fertile. To gain insight into factors that may act redundantly with BMK-1 in spindle assembly and to identify possible additional cellular pathways involving BMK-1, we performed a synthetic lethal screen using the bmk-1 deletion allele ok391. We successfully knocked down 82% of the C. elegans genome using RNAi and assayed viability in bmk-1(ok391) and wild type strains using an automated high-throughput approach based on fluorescence microscopy. The dataset includes a final list of 37 synthetic lethal interactions whose further study is likely to provide insight into kinesin-5 function. PMID:25984351

  13. Crystal structure of F-93 from Sulfolobus spindle-shaped virus 1, a winged-helix DNA binding protein.

    PubMed

    Kraft, Paul; Oeckinghaus, Andrea; Kümmel, Daniel; Gauss, George H; Gilmore, John; Wiedenheft, Blake; Young, Mark; Lawrence, C Martin

    2004-11-01

    Sulfolobus spindle-shaped viruses (SSVs), or Fuselloviridae, are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world (pH /=70 degrees C). Because they are relatively easy to isolate, they represent the best studied of the crenarchaeal viruses. This is particularly true for the type virus, SSV1, which contains a double-stranded DNA genome of 15.5 kilobases, encoding 34 putative open reading frames. Interestingly, the genome shows little sequence similarity to organisms other than its SSV homologues. Together, sequence similarity and biochemical analyses have suggested functions for only 6 of the 34 open reading frames. Thus, even though SSV1 is the best-studied crenarchaeal virus, functions for most (28) of its open reading frames remain unknown. We have undertaken biochemical and structural studies for the gene product of open reading frame F-93. We find that F-93 exists as a homodimer in solution and that a tight dimer is also present in the 2.7-A crystal structure. Further, the crystal structure reveals a fold that is homologous to the SlyA and MarR subfamilies of winged-helix DNA binding proteins. This strongly suggests that F-93 functions as a transcription factor that recognizes a (pseudo-)palindromic DNA target sequence.

  14. Crystal Structure of F-93 from Sulfolobus Spindle-Shaped Virus 1, a Winged-Helix DNA Binding Protein

    PubMed Central

    Kraft, Paul; Oeckinghaus, Andrea; Kümmel, Daniel; Gauss, George H.; Gilmore, John; Wiedenheft, Blake; Young, Mark; Lawrence, C. Martin

    2004-01-01

    Sulfolobus spindle-shaped viruses (SSVs), or Fuselloviridae, are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world (pH ≤4.0; temperature of ≥70°C). Because they are relatively easy to isolate, they represent the best studied of the crenarchaeal viruses. This is particularly true for the type virus, SSV1, which contains a double-stranded DNA genome of 15.5 kilobases, encoding 34 putative open reading frames. Interestingly, the genome shows little sequence similarity to organisms other than its SSV homologues. Together, sequence similarity and biochemical analyses have suggested functions for only 6 of the 34 open reading frames. Thus, even though SSV1 is the best-studied crenarchaeal virus, functions for most (28) of its open reading frames remain unknown. We have undertaken biochemical and structural studies for the gene product of open reading frame F-93. We find that F-93 exists as a homodimer in solution and that a tight dimer is also present in the 2.7-Å crystal structure. Further, the crystal structure reveals a fold that is homologous to the SlyA and MarR subfamilies of winged-helix DNA binding proteins. This strongly suggests that F-93 functions as a transcription factor that recognizes a (pseudo-)palindromic DNA target sequence. PMID:15479795

  15. Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo.

    PubMed

    Laycock, Joseph E; Savoian, Matthew S; Glover, David M

    2006-06-01

    The metaphase-spindle steady-state length occurs as spindle microtubules ;flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance.

  16. Transport of c-MYC by Kinesin-1 for proteasomal degradation in the cytoplasm.

    PubMed

    Lee, Clement M

    2014-09-01

    c-MYC is an oncogenic transcription factor that is degraded by the proteasome pathway. However, the mechanism that regulates delivery of c-MYC to the proteasome for degradation is not well characterized. Here, the results show that the motor protein complex Kinesin-1 transports c-MYC to the cytoplasm for proteasomal degradation. Inhibition of Kinesin-1 function enhanced ubiquitination of c-MYC and induced aggregation of c-MYC in the cytoplasm. Transport studies showed that the c-MYC aggregates moved from the nucleus to the cytoplasm and KIF5B is responsible for the transport in the cytoplasm. Furthermore, inhibition of the proteasomal degradation process also resulted in an accumulation of c-MYC aggregates in the cytoplasm. Moreover, Kinesin-1 was shown to interact with c-MYC and the proteasome subunit S6a. Inhibition of Kinesin-1 function also reduced c-MYC-dependent transformation activities. Taken together, the results strongly suggest that Kinesin-1 transports c-MYC for proteasomal degradation in the cytoplasm and the proper degradation of c-MYC mediated by Kinesin-1 transport is important for transformation activities of c-MYC. In addition, the results indicate that Kinesin-1 transport mechanism is important for degradation of a number of other proteins as well. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Conventional kinesin: Biochemical heterogeneity and functional implications in health and disease.

    PubMed

    Morfini, Gerardo; Schmidt, Nadine; Weissmann, Carina; Pigino, Gustavo; Kins, Stefan

    2016-09-01

    Intracellular trafficking events powered by microtubule-based molecular motors facilitate the targeted delivery of selected molecular components to specific neuronal subdomains. Within this context, we provide a brief review of mechanisms underlying the execution of axonal transport (AT) by conventional kinesin, the most abundant kinesin-related motor protein in the mature nervous system. We emphasize the biochemical heterogeneity of this multi-subunit motor protein, further discussing its significance in light of recent discoveries revealing its regulation by various protein kinases. In addition, we raise issues relevant to the mode of conventional kinesin attachment to cargoes and examine recent evidence linking alterations in conventional kinesin phosphorylation to the pathogenesis of adult-onset neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The von Hippel-Lindau tumour suppressor interacts with microtubules through kinesin-2.

    PubMed

    Lolkema, Martijn P; Mans, Dorus A; Snijckers, Cristel M; van Noort, Mascha; van Beest, Moniek; Voest, Emile E; Giles, Rachel H

    2007-10-02

    Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, however little is known how this occurs. We have identified the kinesin-2 motor complex, known to regulate cilia, as a novel and endogenous pVHL binding partner. The interaction with kinesin-2 facilitates pVHL binding to microtubules. These data suggest that microtubule-dependent functions of pVHL are influenced by kinesin-2.

  19. Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility

    SciTech Connect

    Gearhart, Debra A. . E-mail: dgearhar@mcg.edu; Sickles, Dale W.; Buccafusco, Jerry J.; Prendergast, Mark A.; Terry, Alvin V.

    2007-01-01

    Diisopropylfluorophosphate, originally developed as a chemical warfare agent, is structurally similar to nerve agents, and chlorpyrifos has extensive worldwide use as an agricultural pesticide. While inhibition of cholinesterases underlies the acute toxicity of these organophosphates, we previously reported impaired axonal transport in the sciatic nerves from rats treated chronically with subthreshold doses of chlorpyrifos. Those data indicate that chlorpyrifos (and/or its active metabolite, chlorpyrifos-oxon) might directly affect the function of kinesin and/or microtubules-the principal proteins that mediate anterograde axonal transport. The current report describes in vitro assays to assess the concentration-dependent effects of chlorpyrifos (0-10 {mu}M), chlorpyrifos-oxon (0-10 {mu}M), and diisopropylfluorophosphate (0-0.59 nM) on kinesin-dependent microtubule motility. Preincubating bovine brain microtubules with the organophosphates did not alter kinesin-mediated microtubule motility. In contrast, preincubation of bovine brain kinesin with diisopropylfluorophosphate, chlorpyrifos, or chlorpyrifos-oxon produced a concentration-dependent increase in the number of locomoting microtubules that detached from the kinesin-coated glass cover slip. Our data suggest that the organophosphates-chlorpyrifos-oxon, chlorpyrifos, and diisopropylfluorophosphate-directly affect kinesin, thereby disrupting kinesin-dependent transport on microtubules. Kinesin-dependent movement of vesicles, organelles, and other cellular components along microtubules is fundamental to the organization of all eukaryotic cells, especially in neurons where organelles and proteins synthesized in the cell body must move down long axons to pre-synaptic sites in nerve terminals. We postulate that disruption of kinesin-dependent intracellular transport could account for some of the long-term effects of organophosphates on the peripheral and central nervous system.

  20. The Nek6 and Nek7 protein kinases are required for robust mitotic spindle formation and cytokinesis.

    PubMed

    O'Regan, Laura; Fry, Andrew M

    2009-07-01

    Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

  1. Structural insights into human Kif7, a kinesin involved in Hedgehog signalling

    SciTech Connect

    Klejnot, Marta Kozielski, Frank

    2012-02-01

    The human Kif7 motor domain structure provides insights into a kinesin of medical significance. Kif7, a member of the kinesin 4 superfamily, is implicated in a variety of diseases including Joubert, hydrolethalus and acrocallosal syndromes. It is also involved in primary cilium formation and the Hedgehog signalling pathway and may play a role in cancer. Its activity is crucial for embryonic development. Kif7 and Kif27, a closely related kinesin in the same subfamily, are orthologues of the Drosophila melano@@gaster kinesin-like protein Costal-2 (Cos2). In vertebrates, they work together to fulfil the role of the single Cos2 gene in Drosophila. Here, the high-resolution structure of the human Kif7 motor domain is reported and is compared with that of conventional kinesin, the founding member of the kinesin superfamily. These data are a first step towards structural characterization of a kinesin-4 family member and of this interesting molecular motor of medical significance.

  2. Aluminum-induced kinesin inactivation as potential molecular cause of impairment of neuronal transport processes.

    PubMed

    Böhm, Konrad J; Shabanpour, Mitra; Kalchishkova, Nikolina

    2015-06-15

    It is commonly accepted that aluminum ions may initiate the development of diverse diseases, including neurological disorders. So far, our knowledge of the molecular mechanisms of the interaction of aluminum with defined cellular structures has been still fragmentary. As functional key tasks of neuronal cells essentially depend on the activity of kinesin, we wanted to find out whether this motor protein represents a molecular target for aluminum. We demonstrate that aluminum ions inhibit (IC50 ∼50 μM) the ATPase of the neuron-specific kinesin KIF5A. The ATPase-active center itself, which is located in the kinesin motor domain, does not seem to be directly affected by aluminum. Our results suggest that inhibition is preferentially caused by aluminum binding to some sequence within the kinesin stalk leading to a conformational state of the kinesin molecule, similar to those described in cases of kinesin autoinhibition caused by motor domain-tail binding. Because of the relative high sequence conservation of mammalian kinesin-1 (to which KIF5A belongs), we assume that also in non-neuronal cells the intracellular transport can be affected by aluminum ions.

  3. The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement.

    PubMed

    Cao, Luyan; Wang, Weiyi; Jiang, Qiyang; Wang, Chunguang; Knossow, Marcel; Gigant, Benoît

    2014-11-14

    Kinesin-1 is a dimeric ATP-dependent motor protein that moves towards microtubules (+) ends. This movement is driven by two conformations (docked and undocked) of the two motor domains carboxy-terminal peptides (named neck linkers), in correlation with the nucleotide bound to each motor domain. Despite extensive data on kinesin-1, the structural connection between its nucleotide cycle and movement has remained elusive, mostly because the structure of the critical tubulin-bound apo-kinesin state was unknown. Here we report the 2.2 Å structure of this complex. From its comparison with detached kinesin-ADP and tubulin-bound kinesin-ATP, we identify three kinesin motor subdomains that move rigidly along the nucleotide cycle. Our data reveal how these subdomains reorient on binding to tubulin and when ATP binds, leading respectively to ADP release and to neck linker docking. These results establish a framework for understanding the transformation of chemical energy into mechanical work by (+) end-directed kinesins.

  4. Emergent Properties of the Metaphase Spindle

    PubMed Central

    Reber, Simone; Hyman, Anthony A.

    2015-01-01

    A metaphase spindle is a complex structure consisting of microtubules and a myriad of different proteins that modulate microtubule dynamics together with chromatin and kinetochores. A decade ago, a full description of spindle formation and function seemed a lofty goal. Here, we describe how work in the last 10 years combining cataloging of spindle components, the characterization of their biochemical activities using single-molecule techniques, and theory have advanced our knowledge. Taken together, these advances suggest that a full understanding of spindle assembly and function may soon be possible. PMID:26134313

  5. Centrosomal protein of 192 kDa (Cep192) promotes centrosome-driven spindle assembly by engaging in organelle-specific Aurora A activation

    PubMed Central

    Joukov, Vladimir; De Nicolo, Arcangela; Rodriguez, Alison; Walter, Johannes C.; Livingston, David M.

    2010-01-01

    Centrosomes are primary microtubule (MT)-organizing centers (MTOCs). During mitosis, they dramatically increase their size and MT-nucleating activity and participate in spindle assembly from spindle poles. These events require the serine/threonine kinase, Aurora A (AurA), and the centrosomal protein of 192 kDa (Cep192)/spindle defective 2 (Spd-2), but the underlying mechanism remains unclear. We have found that Cep192, unlike targeting protein for Xklp2 (TPX2), a known MT-localizing AurA activator, is an AurA cofactor in centrosome-driven spindle assembly. Cep192, through a direct interaction, targets AurA to mitotic centrosomes where the locally accumulating AurA forms homodimers or oligomers. The dimerization of endogenous AurA, in the presence of bound Cep192, triggers potent kinase activation that, in turn, drives MT assembly. Depletion of Cep192 or specific interference with AurA-Cep192 binding did not prevent AurA oligomerization on MTs but abrogated AurA recruitment to centrosomes and its activation by either sperm nuclei or anti-AurA antibody (αAurA)-induced dimerization. In these settings, MT assembly by both centrosomes and αAurA-coated beads was also abolished or severely compromised. Hence, Cep192 activates AurA by a mechanism different from that previously described for TPX2. The Cep192-mediated mechanism maximizes AurA activity at centrosomes and appears essential for the function of these organelles as MTOCs. PMID:21097701

  6. Loss of Function of Saccharomyces Cerevisiae Kinesin-Related Cin8 and Kip1 Is Suppressed by Kar3 Motor Domain Mutations

    PubMed Central

    Hoyt, M. A.; He, L.; Totis, L.; Saunders, W. S.

    1993-01-01

    The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-δ strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or ``motor'') domain. These mutations also suppressed the inviability associated with the cin8-δ kip1-δ genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar. PMID:8224825

  7. Clonal Tests of Conventional Kinesin Function during Cell Proliferation and Differentiation

    PubMed Central

    Brendza, Robert P.; Sheehan, Kathy B.; Turner, F.R.; Saxton, William M.

    2000-01-01

    Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks. PMID:10749933

  8. EZH2 is required for mouse oocyte meiotic maturation by interacting with and stabilizing spindle assembly checkpoint protein BubRI

    PubMed Central

    Qu, Yi; Lu, Danyu; Jiang, Hao; Chi, Xiaochun; Zhang, Hongquan

    2016-01-01

    Enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 Lys 27 and plays key roles in a variety of biological processes. Stability of spindle assembly checkpoint protein BubR1 is essential for mitosis in somatic cells and for meiosis in oocytes. However, the role of EZH2 in oocyte meiotic maturation was unknown. Here, we presented a mechanism underlying EZH2 control of BubR1 stability in the meiosis of mouse oocytes. We identified a methyltransferase activity-independent function of EZH2 by demonstrating that EZH2 regulates spindle assembly and the polar body I extrusion. EZH2 was increased with the oocyte progression from GVBD to MII, while EZH2 was concentrated on the chromosomes. Interestingly, inhibition of EZH2 methyltranferase activity by DZNep or GSK343 did not affect oocyte meiotic maturation. However, depletion of EZH2 by morpholino led to chromosome misalignment and abnormal spindle assembly. Furthermore, ectopic expression of EZH2 led to oocyte meiotic maturation arrested at the MI stage followed by chromosome misalignment and aneuploidy. Mechanistically, EZH2 directly interacted with and stabilized BubR1, an effect driving EZH2 into the concert of meiosis regulation. Collectively, we provided a paradigm that EZH2 is required for mouse oocyte meiotic maturation. PMID:27226494

  9. The 14-3-3 protein Bmh1 functions in the spindle position checkpoint by breaking Bfa1 asymmetry at yeast centrosomes.

    PubMed

    Caydasi, Ayse Koca; Micoogullari, Yagmur; Kurtulmus, Bahtiyar; Palani, Saravanan; Pereira, Gislene

    2014-07-15

    In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1-centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation.

  10. The 14-3-3 protein Bmh1 functions in the spindle position checkpoint by breaking Bfa1 asymmetry at yeast centrosomes

    PubMed Central

    Caydasi, Ayse Koca; Micoogullari, Yagmur; Kurtulmus, Bahtiyar; Palani, Saravanan; Pereira, Gislene

    2014-01-01

    In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1–centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation. PMID:24850890

  11. Traffic control: regulation of kinesin motors.

    PubMed

    Verhey, Kristen J; Hammond, Jennetta W

    2009-11-01

    Kinesins are a family of molecular motors that use the energy of ATP hydrolysis to move along the surface of, or destabilize, microtubule filaments. Much progress has been made in understanding the mechanics and functions of the kinesin motors that play important parts in cell division, cell motility, intracellular trafficking and ciliary function. How kinesins are regulated in cells to ensure the temporal and spatial fidelity of their microtubule-based activities is less well understood. Recent work has revealed molecular mechanisms that control kinesin autoinhibition and subsequent activation, binding to cargos and microtubule tracks, and localization at specific sites of action.

  12. Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2

    PubMed Central

    Gable, Alyssa; Qiu, Minhua; Titus, Janel; Balchand, Sai; Ferenz, Nick P.; Ma, Nan; Collins, Elizabeth S.; Fagerstrom, Carey; Ross, Jennifer L.; Yang, Ge; Wadsworth, Patricia

    2012-01-01

    Kinesin-5 is an essential mitotic motor. However, how its spatial–temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification–tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end–directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end–directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2. PMID:22337772

  13. Mechanistic Analysis of the Mitotic Kinesin Eg5*

    PubMed Central

    Cochran, Jared C.; Sontag, Christopher A.; Maliga, Zoltan; Kapoor, Tarun M.; Correia, John J.; Gilbert, Susan P.

    2005-01-01

    Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: kcat = 5.5 s−1, K1/2,Mt = 0.7 μm, and Km,ATP = 25 μm; and for Eg5-437: kcat = 2.9 s−1, K1/2,Mt = 4.5 μm, and Km,ATP = 19 μm. 2′(3′)-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2–3 μm−1s−1, followed immediately by ATP hydrolysis at 15 s−1. ATP-dependent Mt·Eg5 dissociation was relatively slow and rate-limiting at 8 s−1 with mantADP release at 40 s−1. Surprisingly, Eg5-367 binds microtubules more effectively (11 μm−1s−1) than Eg5-437 (0.7 −m−1s−1), consistent with the steady-state K1/2,Mt and the mantADP release K1/2,Mt. These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover. PMID:15247293

  14. Effects of Obstacles on the Dynamics of Kinesins, Including Velocity and Run Length, Predicted by a Model of Two Dimensional Motion

    PubMed Central

    Nam, Woochul; Epureanu, Bogdan I.

    2016-01-01

    Kinesins are molecular motors which walk along microtubules by moving their heads to different binding sites. The motion of kinesin is realized by a conformational change in the structure of the kinesin molecule and by a diffusion of one of its two heads. In this study, a novel model is developed to account for the 2D diffusion of kinesin heads to several neighboring binding sites (near the surface of microtubules). To determine the direction of the next step of a kinesin molecule, this model considers the extension in the neck linkers of kinesin and the dynamic behavior of the coiled-coil structure of the kinesin neck. Also, the mechanical interference between kinesins and obstacles anchored on the microtubules is characterized. The model predicts that both the kinesin velocity and run length (i.e., the walking distance before detaching from the microtubule) are reduced by static obstacles. The run length is decreased more significantly by static obstacles than the velocity. Moreover, our model is able to predict the motion of kinesin when other (several) motors also move along the same microtubule. Furthermore, it suggests that the effect of mechanical interaction/interference between motors is much weaker than the effect of static obstacles. Our newly developed model can be used to address unanswered questions regarding degraded transport caused by the presence of excessive tau proteins on microtubules. PMID:26808534

  15. Small Crowders Slow Down Kinesin-1 Stepping by Hindering Motor Domain Diffusion

    NASA Astrophysics Data System (ADS)

    Sozański, Krzysztof; Ruhnow, Felix; Wiśniewska, Agnieszka; Tabaka, Marcin; Diez, Stefan; Hołyst, Robert

    2015-11-01

    The dimeric motor protein kinesin-1 moves processively along microtubules against forces of up to 7 pN. However, the mechanism of force generation is still debated. Here, we point to the crucial importance of diffusion of the tethered motor domain for the stepping of kinesin-1: small crowders stop the motor at a viscosity of 5 m P a .s —corresponding to a hydrodynamic load in the sub-fN (˜1 0-4 pN ) range—whereas large crowders have no impact even at viscosities above 100 m P a .s . This indicates that the scale-dependent, effective viscosity experienced by the tethered motor domain is a key factor determining kinesin's functionality. Our results emphasize the role of diffusion in the kinesin-1 stepping mechanism and the general importance of the viscosity scaling paradigm in nanomechanics.

  16. Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

    DOE PAGES

    VanDelinder, Virginia; Adams, Peter G.; Bachand, George D.

    2016-12-21

    The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continuemore » to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.« less

  17. Small Crowders Slow Down Kinesin-1 Stepping by Hindering Motor Domain Diffusion.

    PubMed

    Sozański, Krzysztof; Ruhnow, Felix; Wiśniewska, Agnieszka; Tabaka, Marcin; Diez, Stefan; Hołyst, Robert

    2015-11-20

    The dimeric motor protein kinesin-1 moves processively along microtubules against forces of up to 7 pN. However, the mechanism of force generation is still debated. Here, we point to the crucial importance of diffusion of the tethered motor domain for the stepping of kinesin-1: small crowders stop the motor at a viscosity of 5 mPa·s-corresponding to a hydrodynamic load in the sub-fN (~10^{-4} pN) range-whereas large crowders have no impact even at viscosities above 100 mPa·s. This indicates that the scale-dependent, effective viscosity experienced by the tethered motor domain is a key factor determining kinesin's functionality. Our results emphasize the role of diffusion in the kinesin-1 stepping mechanism and the general importance of the viscosity scaling paradigm in nanomechanics.

  18. Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

    SciTech Connect

    VanDelinder, Virginia; Adams, Peter G.; Bachand, George D.

    2016-12-21

    The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.

  19. Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

    PubMed Central

    VanDelinder, Virginia; Adams, Peter G.; Bachand, George D.

    2016-01-01

    The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation. PMID:28000714

  20. Insights into the micromechanical properties of the metaphase spindle

    PubMed Central

    Shimamoto, Yuta; Maeda, Yusuke T.; Ishiwata, Shin'ichi; Libchaber, Albert J.; Kapoor, Tarun M.

    2011-01-01

    SUMMARY The microtubule-based metaphase spindle is subjected to forces that act in diverse orientations and over a wide-range of timescales. Currently, we cannot explain how this dynamic structure generates and responds to forces while maintaining overall stability, as we have a poor understanding of its micromechanical properties. Here we combine the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations to analyze the vertebrate metaphase spindle's timescale- and orientation-dependent viscoelastic properties. We find that spindle viscosity depends on microtubule crosslinking and density. Spindle elasticity can be linked to kinetochore and non-kinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. These data suggest a quantitative model for the micromechanics of this cytoskeletal architecture and provide insight into how structural and functional stability is maintained in the face of forces, such as those that control spindle size and position, and can result from deformations associated with chromosome movement. PMID:21703450

  1. Samurai sword sets spindle size.

    PubMed

    Reber, Simone; Hyman, Anthony A

    2011-12-09

    Although the parts list is nearly complete for many cellular structures, mechanisms that control their size remain poorly understood. Loughlin and colleagues now show that phosphorylation of a single residue of katanin, a microtubule-severing protein, largely accounts for the difference in spindle length between two closely related frogs.

  2. Molecular wear of microtubules propelled by surface-adhered kinesins

    NASA Astrophysics Data System (ADS)

    Dumont, Emmanuel L. P.; Do, Catherine; Hess, Henry

    2015-02-01

    Wear is the progressive loss of material from a body caused by contact and relative movement and is a major concern in both engineering and biology. Advances in nanotechnology have allowed the origins of wear processes to be studied at the atomic and molecular scale, but also demand that wear in nanoscale systems can be predicted and controlled. Biomolecular systems can undergo a range of active movements at the nanoscale, which are enabled by the transduction of chemical energy into mechanical work by polymerization processes and motor proteins. The active movements are accompanied by dissipative processes that can be conceptually understood as ‘protein friction’. Here, we show that wear also occurs in an in vitro system consisting of microtubules gliding across a surface coated with kinesin-1 motor proteins, and that energetic considerations suggest a molecule-by-molecule removal of tubulin proteins. The rates of removal show a complex dependence on sliding velocity and kinesin density, which, in contrast to the friction behaviour between microtubules and kinesin-8, cannot be explained by simple chemical reaction kinetics.

  3. Molecular crowding creates traffic jams of kinesin motors on microtubules

    PubMed Central

    Leduc, Cécile; Padberg-Gehle, Kathrin; Varga, Vladimír; Helbing, Dirk; Diez, Stefan; Howard, Jonathon

    2012-01-01

    Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions density- and bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionarily adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends. PMID:22431622

  4. Molecular crowding creates traffic jams of kinesin motors on microtubules.

    PubMed

    Leduc, Cécile; Padberg-Gehle, Kathrin; Varga, Vladimír; Helbing, Dirk; Diez, Stefan; Howard, Jonathon

    2012-04-17

    Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions density- and bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionarily adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends.

  5. Rho of Plant GTPase Signaling Regulates the Behavior of Arabidopsis Kinesin-13A to Establish Secondary Cell Wall Patterns[W

    PubMed Central

    Oda, Yoshihisa; Fukuda, Hiroo

    2013-01-01

    Plant cortical microtubule arrays determine the cell wall deposition pattern and proper cell shape and function. Although various microtubule-associated proteins regulate the cortical microtubule array, the mechanisms underlying marked rearrangement of cortical microtubules during xylem differentiation are not fully understood. Here, we show that local Rho of Plant (ROP) GTPase signaling targets an Arabidopsis thaliana kinesin-13 protein, Kinesin-13A, to cortical microtubules to establish distinct patterns of secondary cell wall formation in xylem cells. Kinesin-13A was preferentially localized with cortical microtubules in secondary cell wall pits, areas where cortical microtubules are depolymerized to prevent cell wall deposition. This localization of Kinesin-13A required the presence of the activated ROP GTPase, MICROTUBULE DEPLETION DOMAIN1 (MIDD1) protein, and cortical microtubules. Knockdown of Kinesin-13A resulted in the formation of smaller secondary wall pits, while overexpression of Kinesin-13A enlarged their surface area. Kinesin-13A alone could depolymerize microtubules in vitro; however, both MIDD1 and Kinesin-13A were required for the depolymerization of cortical microtubules in vivo. These results indicate that Kinesin-13A regulates the formation of secondary wall pits by promoting cortical microtubule depolymerization via the ROP-MIDD1 pathway. PMID:24280391

  6. TORC1 kinase and the S-phase cyclin Clb5 collaborate to promote mitotic spindle assembly and DNA replication in S. cerevisiae

    PubMed Central

    Tran, Lieu T.; Wang’ondu, Ruth W.; Weng, Jessica B.; Wanjiku, Grace W.; Fong, Chi M.; Kile, Andrew C.; Koepp, Deanna M.; Hood-DeGrenier, Jennifer K.

    2011-01-01

    The Target of Rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is inhibited by the drug rapamycin. In the budding yeast Saccharomyces cerevisiae, translational defects associated with TORC1 inactivation inhibit cell cycle progression at an early stage in G1, but little is known about the possible roles for TORC1 later in the cell cycle. We investigated the rapamycin-hypersensitivity phenotype of cells lacking the S phase cyclin Clb5 (clb5Δ) as a basis for uncovering novel connections between TORC1 and the cell cycle regulatory machinery. Dosage suppression experiments suggested that the clb5Δ rapamycin hypersensitivity reflects a unique Clb5-associated cyclin-dependent kinase (CDK) function that cannot be performed by mitotic cyclins and that also involves motor proteins, particularly the kinesin-like protein Kip3. Synchronized cell experiments revealed rapamycin-induced defects in pre-anaphase spindle assembly and S phase progression that were more severe in clb5Δ than in wild type cells but no apparent activation of Rad53-dependent checkpoint pathways. Some rapamycin-treated cells had aberrant spindle morphologies, but rapamycin did not cause gross defects in the microtubule cytoskeleton. We propose a model in which TORC1 and Clb5/CDK act coordinately to promote both spindle assembly via a pathway involving Kip3 and S phase progression. PMID:20697716

  7. Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2

    PubMed Central

    Zeng, Kang; Bastos, Ricardo Nunes

    2010-01-01

    Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regulatory subunits interferes with spindle formation and chromosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A–TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A–TPX2 complexes in vitro, whereas catalytically inactive mutants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phosphatase regulating Aurora A activity during spindle formation and suggest the general importance of this form of regulation. PMID:21187329

  8. Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

    PubMed Central

    Villeneuve, Julien; van Galen, Josse; Cruz-Garcia, David; Tagaya, Mitsuo

    2013-01-01

    Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface. PMID:23857769

  9. Effect of fuel concentration on cargo transport by a team of Kinesin motors

    NASA Astrophysics Data System (ADS)

    Takshak, Anjneya; Mishra, Nirvantosh; Kulkarni, Aditi; Kunwar, Ambarish

    2017-02-01

    Eukaryotic cells employ specialized proteins called molecular motors for transporting organelles and vesicles from one location to another in a regulated and directed manner. These molecular motors often work collectively in a team while transporting cargos. Molecular motors use cytoplasmic ATP as fuel, which is hydrolyzed to generate mechanical force. While the effect of ATP concentration on cargo transport by single Kinesin motor function is well understood, it is still unexplored, both theoretically and experimentally, how ATP concentration would affect cargo transport by a team of Kinesin motors. For instance, how does fuel concentration affect the travel distances and travel velocities of cargo? How cooperativity of Kinesin motors engaged on a cargo is affected by ATP concentration? To answer these questions, here we develop mechano-chemical models of cargo transport by a team of Kinesin motors. To develop these models we use experimentally-constrained mechano-chemical model of a single Kinesin motor as well as earlier developed mean-field and stochastic models of load sharing for cargo transport. Thus, our new models for cargo transport by a team of Kinesin motors include fuel concentration explicitly, which was not considered in earlier models. We make several interesting predictions which can be tested experimentally. For instance, the travel distances of cargos are very large at limited ATP concentrations in spite of very small travel velocity. Velocities of cargos driven by multiple Kinesin have a Michaelis-Menten dependence on ATP concentration. Similarly, cooperativity among the engaged Kinesin motors on the cargo shows a Michaelis-Menten type dependence, which attains a maximum value near physiological ATP concentrations. Our new results can be potentially useful in controlling artificial nano-molecular shuttles precisely for targeted delivery in various nano-technological applications.

  10. KLP6: a newly identified kinesin that regulates the morphology and transport of mitochondria in neuronal cells.

    PubMed

    Tanaka, Kousuke; Sugiura, Yoshimi; Ichishita, Ryohei; Mihara, Katsuyoshi; Oka, Toshihiko

    2011-07-15

    Mitochondria utilize diverse cytoskeleton-based mechanisms to control their functions and morphology. Here, we report a role for kinesin-like protein KLP6, a newly identified member of the kinesin family, in mitochondrial morphology and dynamics. An RNA interference screen using Caenorhabditis elegans led us to identify a C. elegans KLP-6 involved in maintaining mitochondrial morphology. We cloned a cDNA coding for a rat homolog of C. elegans KLP-6, which is an uncharacterized kinesin in vertebrates. A rat KLP6 mutant protein lacking the motor domain induced changes in mitochondrial morphology and significantly decreased mitochondrial motility in HeLa cells, but did not affect the morphology of other organelles. In addition, the KLP6 mutant inhibited transport of mitochondria during anterograde movement in differentiated neuro 2a cells. To date, two kinesins, KIF1Bα and kinesin heavy chain (KHC; also known as KIF5) have been shown to be involved in the distribution of mitochondria in neurons. Expression of the kinesin heavy chain/KIF5 mutant prevented mitochondria from entering into neurites, whereas both the KLP6 and KIF1Bα mutants decreased mitochondrial transport in axonal neurites. Furthermore, both KLP6 and KIF1Bα bind to KBP, a KIF1-binding protein required for axonal outgrowth and mitochondrial distribution. Thus, KLP6 is a newly identified kinesin family member that regulates mitochondrial morphology and transport.

  11. Modelling of internal architecture of kinesin nanomotor as a machine language.

    PubMed

    Khataee, H R; Ibrahim, M Y

    2012-09-01

    Kinesin is a protein-based natural nanomotor that transports molecular cargoes within cells by walking along microtubules. Kinesin nanomotor is considered as a bio-nanoagent which is able to sense the cell through its sensors (i.e. its heads and tail), make the decision internally and perform actions on the cell through its actuator (i.e. its motor domain). The study maps the agent-based architectural model of internal decision-making process of kinesin nanomotor to a machine language using an automata algorithm. The applied automata algorithm receives the internal agent-based architectural model of kinesin nanomotor as a deterministic finite automaton (DFA) model and generates a regular machine language. The generated regular machine language was acceptable by the architectural DFA model of the nanomotor and also in good agreement with its natural behaviour. The internal agent-based architectural model of kinesin nanomotor indicates the degree of autonomy and intelligence of the nanomotor interactions with its cell. Thus, our developed regular machine language can model the degree of autonomy and intelligence of kinesin nanomotor interactions with its cell as a language. Modelling of internal architectures of autonomous and intelligent bio-nanosystems as machine languages can lay the foundation towards the concept of bio-nanoswarms and next phases of the bio-nanorobotic systems development.

  12. Active transport of oil droplets along oriented microtubules by kinesin molecular motors.

    PubMed

    Bottier, Céline; Fattaccioli, Jacques; Tarhan, Mehmet C; Yokokawa, Ryuji; Morin, Fabrice O; Kim, Beomjoon; Collard, Dominique; Fujita, Hiroyuki

    2009-06-21

    We demonstrate the active transport of liquid cargos in the form of oil-in-water emulsion droplets loaded on kinesin motor proteins moving along oriented microtubules. We analyze the motility properties of the kinesin motors (velocity and run length) and find that the liquid cargo in the form of oil droplets does not alter the motor function of the kinesin molecules. This work provides a novel method for handling only a few molecules/particles encapsulated inside the oil droplets and represents a key finding for the integration of kinesin-based active transport into nanoscale lab-on-a-chip devices. We also investigate the effect of the diameter of the droplets on the motility properties of the kinesin motors. The velocity is approximately constant irrespective of the diameter of the droplets whereas we highlight a strong increase of the run length when the diameter of the droplets increases. We correlate these results with the number of kinesin motors involved in the transport process and find an excellent agreement between our experimental result and a theoretical model.

  13. Kinesin-related genes from diplomonad, sponge, amphioxus, and cyclostomes: divergence pattern of kinesin family and evolution of giardial membrane-bounded organella.

    PubMed

    Iwabe, Naoyuki; Miyata, Takashi

    2002-09-01

    To understand the question of whether divergence of eukaryotic genes by gene duplications and domain shufflings proceeded gradually or intermittently during evolution, we have cloned and sequenced Giardia lamblia cDNAs encoding kinesins and kinesin-related proteins and have obtained 13 kinesin-related cDNAs, some of which are likely homologs of vertebrate kinesins involved in vesicle transfer to ER, Golgi, and plasma membrane. A phylogenetic tree of the kinesin family revealed that most gene duplications that gave rise to different kinesin subfamilies with distinct functions have been completed before the earliest divergence of extant eukaryotes. This suggests that the complex endomembrane system has arisen very early in eukaryotic evolution, and the diminutive ER and Golgi apparatus recognized in the giardial cells, together with the absence of mitochondria, might be characters acquired secondarily during the evolution of parasitism. To understand the divergence pattern of the kinesin family in the lineage leading to vertebrates, seven more Unc104-related cDNAs have been cloned from sponge, amphioxus, hagfish, and lamprey. The divergence pattern of the animal Unc104/KIF1 subfamily is characterized by two active periods in gene duplication interrupted by a considerably long period of silence, instead of proceeding gradually: animals underwent extensive gene duplications before the parazoan-eumetazoan split. In the early evolution of vertebrates around the cyclostome-gnathostome split, further gene duplications occurred, by which a variety of genes with similar structures over the entire regions were generated. This pattern of divergence is similar to those of animal genes involved in cell-cell communication and developmental control.

  14. Kinesin-4 Functions in Vesicular Transport on Cortical Microtubules and Regulates Cell Wall Mechanics during Cell Elongation in Plants.

    PubMed

    Kong, Zhaosheng; Ioki, Motohide; Braybrook, Siobhan; Li, Shundai; Ye, Zheng-Hua; Julie Lee, Yuh-Ru; Hotta, Takashi; Chang, Anny; Tian, Juan; Wang, Guangda; Liu, Bo

    2015-07-01

    In plants, anisotropic cell expansion depends on cortical microtubules that serve as tracks along which macromolecules and vesicles are transported by the motor kinesins of unknown identities. We used cotton (Gossypium hirsutum) fibers that underwent robust elongation to discover kinesins that are involved in cell elongation and found Gh KINESIN-4A expressed abundantly. The motor was detected by immunofluorescence on vesicle-like structures that were associated with cortical microtubules. In Arabidopsis thaliana, the orthologous motor At KINESIN-4A/FRA1, previously implicated in cellulose deposition during secondary growth in fiber cells, was examined by live-cell imaging in cells expressing the fluorescently tagged functional protein. The motor decorated vesicle-like particles that exhibit a linear movement along cortical microtubules with an average velocity of 0.89 μm/min, which was significantly different from those linked to cellulose biosynthesis. We also discovered that At KINESIN-4A/FRA1 and the related At KINESIN-4C play redundant roles in cell wall mechanics, cell elongation, and the axial growth of various vegetative and reproductive organs, as the loss of At KINESIN-4C greatly enhanced the defects caused by a null mutation at the KINESIN-4A/FRA1 locus. The double mutant displayed a lack of cell wall softening at normal stages of rapid cell elongation. Furthermore, enhanced deposition of arabinose-containing carbohydrate was detected in the kinesin-4 mutants. Our findings established a connection between the Kinesin-4-based transport of cargoes containing non-cellulosic components along cortical microtubules and cell wall mechanics and cell elongation in flowering plants.

  15. Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage.

    PubMed

    Findley, Seth D; Tamanaha, Mio; Clegg, Nigel J; Ruohola-Baker, Hannele

    2003-03-01

    A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. We have characterized a null allele of maelstrom (mael) and shown that Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, we have defined maelstrom as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, we have determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena, aubergine and spindle-E (spn-E), for its nuage localization. Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway.

  16. Live-Cell Analysis of Mitotic Spindle Formation in Taxol-Treated Cells

    PubMed Central

    Hornick, Jessica E.; Bader, Jason R.; Tribble, Emily K.; Trimble, Kayleigh; Breunig, J. Scott; Halpin, Elizabeth S.; Vaughan, Kevin T.; Hinchcliffe, Edward H.

    2009-01-01

    Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-α tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic redistribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 “dynamitin” does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible—but before NEB is complete—results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation. PMID:18481305

  17. Prevention of mammalian DNA reduplication, following the release from the mitotic spindle checkpoint, requires p53 protein, but not p53-mediated transcriptional activity.

    PubMed

    Notterman, D; Young, S; Wainger, B; Levine, A J

    1998-11-26

    The tumor suppressor p53 has been identified as a component of a mitotic spindle checkpoint. When exposed to a spindle-disrupting drug such as nocodazole, fibroblasts derived from mice having wild-type p53 are blocked with a 4N content of DNA. Conversely, fibroblasts from p53-deficient mice become polyploid. To learn if transcriptional activation of downstream genes by p53 plays a role in this putative checkpoint, three cell lines were exposed to nocodazole. In one line, p53 protein is not expressed, while the other two cell lines over-express p53. In one of these two lines, the N-terminal transactivation domain is wild-type and in the second, this region contains a mutation that eliminates the ability of the protein to act as a transcription factor. Incubation with nocodazole of cells containing wild-type p53 results in accumulation of both 2N and 4N populations of cells. Under the same conditions, cells containing a transactivation-deficient mutant of p53 accumulate a 4N population of cells, but not a 2N population of cells. Cells entirely deficient in p53 protein become hyperdiploid, and display 8N to 16N DNA content. In all three cell lines, nocodazole elicited an initial increase in mitotic cells, but within 24 h the mitotic index returned to baseline. Expression patterns of cyclins B and D indicated that following entry into mitosis, the cells returned to a G1 state but with 4N DNA content. Subsequent re-duplication of DNA beyond 4N is prevented in cells containing either wild-type or transcriptionally inactive p53 protein. In cells entirely lacking p53 protein, DNA is re-duplicated (without an intervening mitosis) and the cells become hyperdiploid. These experiments indicate that p53 does not participate in the transient mitotic arrest that follows spindle disruption, but is essential to prevent subsequent reduplication of DNA and the resulting hyperdiploid state. This function is intact in a mutant that is transcriptionally inactive.

  18. A Functional Mitotic Spindle Prepared from Mammalian Cells in Culture

    PubMed Central

    Cande, W. Zacheus; Snyder, Judith; Smith, Diana; Summers, Keith; McIntosh, J. R.

    1974-01-01

    Mitotic cells lysed into solutions of polymerizable microtubule protein contain a spindle which is similar to the living spindle in two respects: it will lose and gain birefringence when cooled and warmed, and it will move anaphase chromosomes to the opposite ends of the cell. Early anaphase cells lysed into buffers containing high molecular weight polyethylene glycol and nucleotide triphosphates will continue chromosome motion and spindle elongation in the absence of exogenous spindle subunits. These results suggest that while spindle growth requires microtubule polymerization, anaphase motions do not. Images PMID:4524659

  19. Cloning and characterization of Kin5, a novel Tetrahymena ciliary kinesin II.

    PubMed

    Awan, Aashir; Bernstein, Mitchell; Hamasaki, Toshikazu; Satir, Peter

    2004-05-01

    Two Tetrahymena kinesin-like proteins (klps) of the kinesin II subfamily, Kin1 and Kin2, were first identified by Brown et al. [1999: Mol Biol Cell 10: 3081-3096] and shown to be involved in ciliary morphogenesis probably as molecular motors in intraciliary transport (ICT). Using Tetrahymena genomic DNA as a template, we cloned Kin5, another kinesin II subfamily member. Kin5 is upregulated upon deciliation, suggesting that Kin5 is a ciliary protein. Kin5 is most closely related to Osm3, a Caenorhabditis elegans kinesin II; Osm3 and Kin5 have a 56% identity, which rises to 60.4% in the motor domain and a 45% identity in a 60 amino acid region of the C-terminal FERM (4.1, Ezrin, Radixin, Moesin) domain, not present in Kin1 or Kin2, which we hypothesize to be a critical domain either for dimerization or for cargo recognition in ICT. An antibody to a peptide sequence from the tail region of Kin5 localizes in a punctate pattern along the ciliary axoneme, colocalizing with an antibody to the raft protein IFT139. These findings suggest that Kin5 is an ICT motor like Osm3. Osm3 orthologs apparently transport membrane proteins and Kin5 may be the homodimeric kinesin II that performs this function in Tetrahymena cilia. Copyright 2004 Wiley-Liss, Inc.

  20. Long astral microtubules uncouple mitotic spindles from the cytokinetic furrow

    PubMed Central

    Rankin, Kathleen E.

    2010-01-01

    Astral microtubules (MTs) are known to be important for cleavage furrow induction and spindle positioning, and loss of astral MTs has been reported to increase cortical contractility. To investigate the effect of excess astral MT activity, we depleted the MT depolymerizer mitotic centromere-associated kinesin (MCAK) from HeLa cells to produce ultra-long, astral MTs during mitosis. MCAK depletion promoted dramatic spindle rocking in early anaphase, wherein the entire mitotic spindle oscillated along the spindle axis from one proto-daughter cell to the other, driven by oscillations of cortical nonmuscle myosin II. The effect was phenocopied by taxol treatment. Live imaging revealed that cortical actin partially vacates the polar cortex in favor of the equatorial cortex during anaphase. We propose that this renders the polar actin cortex vulnerable to rupture during normal contractile activity and that long astral MTs enlarge the blebs. Excessively large blebs displace mitotic spindle position by cytoplasmic flow, triggering the oscillations as the blebs resolve. PMID:20603328

  1. Poleward microtubule flux mitotic spindles assembled in vitro

    PubMed Central

    1991-01-01

    In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase. PMID:1999464

  2. Characterization and expression profiling of cucumber kinesin genes during early fruit development: revealing the roles of kinesins in exponential cell production and enlargement in cucumber fruit

    PubMed Central

    Yang, Xue Yong; Wang, Yan; Jiang, Wei Jie

    2013-01-01

    Rapid cell division and expansion in early fruit development are important phases for cucumber fruit yield and quality. Kinesin proteins are microtubule-based motors responsible for modulating cell division and enlargement. In this work, the candidate kinesin genes involved in rapid cell division and expansion during cucumber fruit development were investigated. The morphological and cellular changes during early fruit development were compared in four cucumber genotypes with varied fruit size. The correlation between the expression profiles of cucumber kinesin genes and cellular changes in fruit was investigated. Finally, the biochemical characteristics and subcellular localizations of three candidate kinesins were studied. The results clarified the morphological and cellular changes during early cucumber fruit development. This study found that CsKF2–CsKF6 were positively correlated with rapid cell production; CsKF1 and CsKF7 showed a strongly positive correlation with rapid cell expansion. The results also indicated that CsKF1 localized to the plasma membrane of fast-expanding fruit cells, that CsKF2 might play a role in fruit chloroplast division, and that CsKF3 is involved in the function or formation of phragmoplasts in fruit telophase cells. The results strongly suggest that specific fruit-enriched kinesins are specialized in their functions in rapid cell division and expansion during cucumber fruit development. PMID:24023249

  3. Characterization and expression profiling of cucumber kinesin genes during early fruit development: revealing the roles of kinesins in exponential cell production and enlargement in cucumber fruit.

    PubMed

    Yang, Xue Yong; Wang, Yan; Jiang, Wei Jie; Liu, Xiao Ling; Zhang, Xiao Meng; Yu, Hong Jun; Huang, San Wen; Liu, Guo Qin

    2013-11-01

    Rapid cell division and expansion in early fruit development are important phases for cucumber fruit yield and quality. Kinesin proteins are microtubule-based motors responsible for modulating cell division and enlargement. In this work, the candidate kinesin genes involved in rapid cell division and expansion during cucumber fruit development were investigated. The morphological and cellular changes during early fruit development were compared in four cucumber genotypes with varied fruit size. The correlation between the expression profiles of cucumber kinesin genes and cellular changes in fruit was investigated. Finally, the biochemical characteristics and subcellular localizations of three candidate kinesins were studied. The results clarified the morphological and cellular changes during early cucumber fruit development. This study found that CsKF2-CsKF6 were positively correlated with rapid cell production; CsKF1 and CsKF7 showed a strongly positive correlation with rapid cell expansion. The results also indicated that CsKF1 localized to the plasma membrane of fast-expanding fruit cells, that CsKF2 might play a role in fruit chloroplast division, and that CsKF3 is involved in the function or formation of phragmoplasts in fruit telophase cells. The results strongly suggest that specific fruit-enriched kinesins are specialized in their functions in rapid cell division and expansion during cucumber fruit development.

  4. Kinesin-1 inhibits the aggregation of amyloid-β peptide as detected by fluorescence cross-correlation spectroscopy.

    PubMed

    Zheng, Yanpeng; Tian, Shijun; Peng, Xianglei; Yang, Jingfa; Fu, Yuanhui; Jiao, Yueying; Zhao, Jiang; He, Jinsheng; Hong, Tao

    2016-04-01

    Although the exact etiology and pathogenesis of Alzheimer's disease (AD) are still unclear, amyloid-β (Aβ) generated by the proteolytic processing of amyloid-β precursor protein (APP) aggregate to form toxic amyloid species. Kinesin-1 is the first identified ATP-dependent axonal transport motor protein that has been proven to affect Aβ generation and deposition. In this paper, we applied dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) to investigate the direct interaction of Aβ with kinesin-1 at the single-molecule fluorescence level in vitro. The results showed that two kinds of enhanced green fluorescent protein (EGFP)-tagged kinesin light-chain subunits of kinesin-1(KLCs), KLC-E and E-KLC inhibited the aggregation of Aβ over a period of time, providing additional insight into the mechanism of axonal transport deficits in AD.

  5. Opposing motor activities are required for the organization of the mammalian mitotic spindle pole

    PubMed Central

    1996-01-01

    We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end- directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors. PMID:8896597

  6. Mitotic Spindle Proteomics in Chinese Hamster Ovary Cells

    PubMed Central

    Bonner, Mary Kate; Poole, Daniel S.; Xu, Tao; Sarkeshik, Ali; Yates, John R.; Skop, Ahna R.

    2011-01-01

    Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells. PMID:21647379

  7. Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons.

    PubMed

    Terada, S; Kinjo, M; Hirokawa, N

    2000-09-29

    Slow axonal transport depends on an active mechanism that conveys cytosolic proteins. To investigate its molecular mechanism, we now constructed an in vitro experimental system for observation of tubulin transport, using squid giant axons. After injecting fluorescence-labeled tubulin into the axons, we monitored the movement of fluorescence by confocal laser scanning microscopy and fluorescence correlation spectroscopy. Here, from the pharmacological experiments and the functional blocking of kinesin motor protein by anti-kinesin antibody, we show that the directional movement of fluorescent profile was dependent on kinesin motor function. The fluorescent correlation function and estimated translational diffusion time revealed that tubulin molecule was transported in a unique form of large transporting complex distinct from those of stable polymers or other cytosolic protein.

  8. A genomic toolkit to investigate kinesin and myosin motor function in cells.

    PubMed

    Maliga, Zoltan; Junqueira, Magno; Toyoda, Yusuke; Ettinger, Andreas; Mora-Bermúdez, Felipe; Klemm, Robin W; Vasilj, Andrej; Guhr, Elaine; Ibarlucea-Benitez, Itziar; Poser, Ina; Bonifacio, Ezio; Huttner, Wieland B; Shevchenko, Andrej; Hyman, Anthony A

    2013-03-01

    Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

  9. KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly.

    PubMed

    Connolly, Amy A; Sugioka, Kenji; Chuang, Chien-Hui; Lowry, Joshua B; Bowerman, Bruce

    2015-09-14

    During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere-associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(-) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(-) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore-microtubule (k-MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(-) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k-MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.

  10. KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

    PubMed Central

    Connolly, Amy A.; Sugioka, Kenji; Chuang, Chien-Hui; Lowry, Joshua B.

    2015-01-01

    During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly. PMID:26370499

  11. Microscale transport and sorting by kinesin molecular motors.

    PubMed

    Jia, Lili; Moorjani, Samira G; Jackson, Thomas N; Hancock, William O

    2004-03-01

    As biomolecular detection systems shrink in size, there is an increasing demand for systems that transport and position materials at micron- and nanoscale dimensions. Our goal is to combine cellular transport machinery-kinesin molecular motors and microtubules-with integrated optoelectronics into a hybrid biological/engineered microdevice that will bind, transport, and detect specific proteins, DNA/RNA molecules, viruses, or cells. For microscale transport, 1.5 microm deep channels were created with SU-8 photoresist on glass, kinesin motors adsorbed to the bottom of the channels, and the channel walls used to bend and redirect microtubules moving over the immobilized motors. Novel channel geometries were investigated as a means to redirect and sort microtubules moving in these channels. We show that DC and AC electric fields are sufficient to transport microtubules in solution, establishing an approach for redirecting microtubules moving in channels. Finally, we inverted the geometry to demonstrate that kinesins can transport gold nanowires along surface immobilized microtubules, providing a model for nanoscale directed assembly.

  12. A coordinated molecular 'fishing' mechanism in heterodimeric kinesin

    NASA Astrophysics Data System (ADS)

    Hou, Ruizheng; Wang, Zhisong

    2010-09-01

    Kar3 is a kinesin motor that facilitates chromosome segregation during cell division. Unlike many members of the kinesin superfamily, Kar3 forms a heterodimer with non-motor protein Vik1 or Cik1 in vivo. The heterodimers show ATP-driven minus-end directed motility along a microtubule (MT) lattice, and also serve as depolymerase at the MT ends. The molecular mechanisms behind this dual functionality remain mysterious. Here, a molecular mechanical model for the Kar3/Vik1 heterodimer based on structural, kinetic and motility data reveals a long-range chemomechanical transmission mechanism that resembles a familiar fishing tactic. By this molecular 'fishing', ATP-binding to Kar3 dissociates catalytically inactive Vik1 off MT to facilitate minus-end sliding of the dimer on the MT lattice. When the dimer binds the frayed ends of MT, the fishing channels ATP hydrolysis energy into MT deploymerization by a mechanochemical effect. The molecular fishing thus provides a unified mechanistic ground for Kar3's dual functionality. The fishing-promoted depolymerization differs from the depolymerase mechanisms found in homodimeric kinesins. The fishing also enables intermolecular coordination with a chemomechanical coupling feature different from the paradigmatic pattern of homodimeric motors. This study rationalizes some puzzling experimental observation, and suggests new experiments for further elucidation of the fishing mechanism.

  13. Measuring mitotic spindle dynamics in budding yeast

    NASA Astrophysics Data System (ADS)

    Plumb, Kemp

    In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

  14. Mechanical model of kinesin moving on microtubule

    NASA Astrophysics Data System (ADS)

    To, Kiwing; Chou, Ya-Chang; Hsiao, Yi-Feng; Chen, Kuan-Hua

    Kinesins are biomolecules that serve as intercellular motors for carrying cellular cargos along microtubules. Although the mechanism of converting the chemical energy of ATP to mechanical work is not fully understood, the motion of a kinesin on a microtubule has been measured and two different mechanisms, namely the ``hand-over-hand'' and ``inchworm'', has been proposed. The particular shape of kinesin and microtubules suggest a possible mechanism for force generation similar to Brownian ratchet. Using a bead chain connected to two heads that are attracted to a vibrated ratchet plate as a scaled up analog of the kinesinmicrotubule system, we manage to simulate both ``handoverhand'' and ``inchworm'' motion [Chou, et. al., Physica A443, 66 (2015)]. In addition, we find that chain, which play the role of the stalk in a kinesin molecule, can also generate force by interacting with the ratchet plate [Chen, et. al. Phys. Rev. E87, 012711 (2013)].

  15. Multiscale method for modeling binding phenomena involving large objects: application to kinesin motor domains motion along microtubules.

    PubMed

    Li, Lin; Alper, Joshua; Alexov, Emil

    2016-03-18

    Many biological phenomena involve the binding of proteins to a large object. Because the electrostatic forces that guide binding act over large distances, truncating the size of the system to facilitate computational modeling frequently yields inaccurate results. Our multiscale approach implements a computational focusing method that permits computation of large systems without truncating the electrostatic potential and achieves the high resolution required for modeling macromolecular interactions, all while keeping the computational time reasonable. We tested our approach on the motility of various kinesin motor domains. We found that electrostatics help guide kinesins as they walk: N-kinesins towards the plus-end, and C-kinesins towards the minus-end of microtubules. Our methodology enables computation in similar, large systems including protein binding to DNA, viruses, and membranes.

  16. Multiscale method for modeling binding phenomena involving large objects: application to kinesin motor domains motion along microtubules

    NASA Astrophysics Data System (ADS)

    Li, Lin; Alper, Joshua; Alexov, Emil

    2016-03-01

    Many biological phenomena involve the binding of proteins to a large object. Because the electrostatic forces that guide binding act over large distances, truncating the size of the system to facilitate computational modeling frequently yields inaccurate results. Our multiscale approach implements a computational focusing method that permits computation of large systems without truncating the electrostatic potential and achieves the high resolution required for modeling macromolecular interactions, all while keeping the computational time reasonable. We tested our approach on the motility of various kinesin motor domains. We found that electrostatics help guide kinesins as they walk: N-kinesins towards the plus-end, and C-kinesins towards the minus-end of microtubules. Our methodology enables computation in similar, large systems including protein binding to DNA, viruses, and membranes.

  17. Structure of D-63 from Sulfolobus Spindle-Shaped Virus 1: Surface Properties of the Dimeric Four-Helix Bundle Suggest an Adaptor Protein Function

    PubMed Central

    Kraft, Paul; Kümmel, Daniel; Oeckinghaus, Andrea; Gauss, George H.; Wiedenheft, Blake; Young, Mark; Lawrence, C. Martin

    2004-01-01

    Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH ≤ 4.0) hot springs (≥70°C) around the world. SSV1 contains a 15.5-kb double-stranded DNA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a DnaA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP2, and VP3). The functions of the remaining 29 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 D-63. The structure reveals a helix-turn-helix motif that dimerizes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the dimer axis and thus exhibits 2-fold symmetry. Two smaller conserved patches, also related by 2-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that D-63 may function as an adaptor protein in macromolecular assembly. PMID:15220417

  18. The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development

    PubMed Central

    Courtois, Aurélien; Schuh, Melina; Ellenberg, Jan

    2012-01-01

    The transition from meiosis to mitosis, classically defined by fertilization, is a fundamental process in development. However, its mechanism remains largely unexplored. In this paper, we report a surprising gradual transition from meiosis to mitosis over the first eight divisions of the mouse embryo. The first cleavages still largely share the mechanism of spindle formation with meiosis, during which the spindle is self-assembled from randomly distributed microtubule-organizing centers (MTOCs) without centrioles, because of the concerted activity of dynein and kinesin-5. During preimplantation development, the number of cellular MTOCs progressively decreased, the spindle pole gradually became more focused, and spindle length progressively scaled down with cell size. The typical mitotic spindle with centrin-, odf2-, kinesin-12–, and CP110-positive centrosomes was established only in the blastocyst. Overall, the transition from meiosis to mitosis progresses gradually throughout the preimplantation stage in the mouse embryo, thus providing a unique system to study the mechanism of centrosome biogenesis in vivo. PMID:22851319

  19. Polo-like kinase 1 regulates the stability of the mitotic centromere-associated kinesin in mitosis.

    PubMed

    Sanhaji, Mourad; Ritter, Andreas; Belsham, Hannah R; Friel, Claire T; Roth, Susanne; Louwen, Frank; Yuan, Juping

    2014-05-30

    Proper bi-orientation of chromosomes is critical for the accurate segregation of chromosomes in mitosis. A key regulator of this process is MCAK, the mitotic centromere-associated kinesin. During mitosis the activity and localization of MCAK are regulated by mitotic key kinases including Plk1 and Aurora B. We show here that S621 in the MCAK's C-terminal domain is the major phosphorylation site for Plk1. This phosphorylation regulates MCAK's stability and facilitates its recognition by the ubiquitin/proteasome dependent APC/C(Cdc20) pathway leading to its D-box dependent degradation in mitosis. While phosphorylation of S621 does not directly affect its microtubule depolymerising activity, loss of Plk1 phosphorylation on S621 indirectly enhances its depolymerization activity in vivo by stabilizing MCAK, leading to an increased level of protein. Interfering with phosphorylation at S621 causes spindle formation defects and chromosome misalignments. Therefore, this study suggests a new mechanism by which Plk1 regulates MCAK: by regulating its degradation and hence controlling its turnover in mitosis.

  20. The Beginning of Kinesin's Force-Generating Cycle Visualized at 9Angstrom Resolution

    SciTech Connect

    Sindelar, Charles V.; Downing, Kenneth H.

    2007-06-20

    We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in ''ejecting'' adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the ''neck linker''). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself.

  1. Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors

    PubMed Central

    Furuta, Ken’ya; Furuta, Akane; Toyoshima, Yoko Y.; Amino, Misako; Oiwa, Kazuhiro; Kojima, Hiroaki

    2013-01-01

    Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up. PMID:23267076

  2. Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors.

    PubMed

    Furuta, Ken'ya; Furuta, Akane; Toyoshima, Yoko Y; Amino, Misako; Oiwa, Kazuhiro; Kojima, Hiroaki

    2013-01-08

    Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

  3. Functional Asymmetry in Kinesin and Dynein Dimers

    PubMed Central

    Rank, Katherine C.; Rayment, Ivan

    2012-01-01

    Active transport along the microtubule lattice is a complex process that involves both the Kinesin and Dynein superfamily of motors. Transportation requires sophisticated regulation much of which occurs through the motor’s tail domain. However, a significant portion of this regulation also occurs through structural changes that arise in the motor and the microtubule upon binding. The most obvious structural change being the manifestation of asymmetry. To a first approximation in solution, kinesin dimers exhibit two-fold symmetry, and microtubules, helical symmetry. The higher symmetries of both the kinesin dimers and microtubule lattice are lost on formation of the kinesin-microtubule complex. Loss of symmetry has functional consequences such as an asymmetric hand-over-hand mechanism in plus-end directed kinesins, asymmetric microtubule binding in the Kinesin-14 family, spatially biased stepping in dynein, and cooperative binding of additional motors to the microtubule. This review focuses on how the consequences of asymmetry affect regulation of motor heads within a dimer, dimers within an ensemble of motors, and suggests how these asymmetries may affect regulation of active transport within the cell. PMID:23066835

  4. Spatial control of microtubule length and lifetime by opposing stabilizing and destabilizing functions of Kinesin-8

    PubMed Central

    Fukuda, Yusuke; Luchniak, Anna; Murphy, Erin R.; Gupta, Mohan L.

    2014-01-01

    Summary Background To function in diverse cellular processes, the dynamic behavior of microtubules (MTs) must be differentially regulated within the cell. In budding yeast, the spindle position checkpoint (SPOC) inhibits mitotic exit in response to mispositioned spindles. To maintain SPOC-mediated anaphase arrest, astral MTs must maintain persistent interactions with and/or extend through the bud neck. However, the molecular mechanisms that ensure the stability of these interactions are not known. Results The presence of a MT extending through and/or interacting with the bud neck is maintained by spatial control of catastrophe and rescue, which extends MT lifetime >25-fold and controls the length of dynamic MTs within the bud compartment. Moreover, the single kinesin-8 motor, Kip3, alternately mediates both catastrophe and rescue of the bud MT. Kip3 accumulates in a length-dependent manner along the lattice of MTs within the bud. Yet, it induces catastrophe spatially near the bud tip. Rather, this accumulation of Kip3 facilitates its association with depolymerizing MT plus-ends, where Kip3 promotes rescue before MTs exit the bud. MT rescue within the bud requires the tail domain of Kip3, whereas the motor domain mediates catastrophe at the bud tip. In vitro, Kip3 exerts both stabilizing and destabilizing effects on reconstituted yeast MTs. Conclusions The kinesin-8 Kip3 is a multifunctional regulator that differentially stabilizes and destabilizes specific MTs. Control over MT catastrophe and rescue by Kip3 defines the length and lifetime of MTs within the bud compartment of cells with mispositioned spindles. This subcellular regulation of MT dynamics is critical to maintain mitotic arrest in response to mispositioned spindles. PMID:25088560

  5. SHCBP1L, a conserved protein in mammals, is predominantly expressed in male germ cells and maintains spindle stability during meiosis in testis.

    PubMed

    Liu, Mingxi; Shi, Xiaodan; Bi, Ye; Qi, Lin; Guo, Xuejiang; Wang, Lei; Zhou, Zuomin; Sha, Jiahao

    2014-06-01

    Male subfertility due to falling sperm counts has become an increasing problem over a short timescale (50-70 years). Recently, bioinformatics analysis of the human testis proteome has revealed the existence of human-testicular-predominantly-expressed-proteins, which are highly associated with spermatogenesis, although the functions of many of these proteins are still unknown. To understand the function of one of these proteins, SHCBP1L (1700012A16RIKEN), a knockout mouse was produced in which this gene was removed. Using this model, we showed that SHCBP1L binds to another protein, HSPA2, and maintains stability of the spindle. We showed that this complex was not present in knockout mice and that an abnormal number of spermatocytes were held in the early stages of meiosis. Many of these cells were undergoing programmed cell-death, or apoptosis, which is highly unusual for cells during the early stages of meiosis. We also found that proteins very similar to SHCBP1L exist in many other mammals. This led us to propose that SHCBP1L plays an important role in spermatogenesis in mammals. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Regulation of mitotic spindle asymmetry by SUMO and the spindle-assembly checkpoint in yeast.

    PubMed

    Leisner, Christian; Kammerer, Daniel; Denoth, Annina; Britschi, Mirjam; Barral, Yves; Liakopoulos, Dimitris

    2008-08-26

    During mitosis, the kinetochore microtubules capture and segregate chromosomes, and the astral microtubules position the spindle within the cell. Although the spindle is symmetric, proper positioning of the spindle in asymmetrically dividing cells generally correlates with the formation of morphologically and structurally distinct asters [1]. In budding yeast, the spindle-orientation proteins Kar9 and dynein decorate only one aster of the metaphase spindle and direct it toward the bud [2, 3]. The mechanisms controlling the distribution of Kar9 and dynein remain unclear. Here, we show that SUMO regulates astral-microtubule function in at least two ways. First, Kar9 was sumoylated in vivo. Sumoylation and Cdk1-dependent phosphorylation of Kar9 independently promoted Kar9 asymmetry on the spindle. Second, proper regulation of kinetochore function by SUMO was also required for Kar9 asymmetry. Indeed, activation of the spindle-assembly checkpoint (SAC) due to SUMO and kinetochore defects promoted symmetric redistribution of Kar9 in a Mad2-dependent manner. The control of Kar9 distribution by the SAC was independent of Kar9 sumoylation and phosphorylation. Together, our data reveal that three independent mechanisms contribute to Kar9 asymmetry: Cdk1-dependent phosphorylation, sumoylation, and SAC signaling. Hence, the two seemingly independent spindle domains, kinetochores and astral microtubules, function in a tightly coordinated fashion.

  7. Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture

    PubMed Central

    Ganguly, Sujoy; Williams, Lucy S.; Palacios, Isabel M.; Goldstein, Raymond E.

    2012-01-01

    Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA. PMID:22949706

  8. Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture.

    PubMed

    Ganguly, Sujoy; Williams, Lucy S; Palacios, Isabel M; Goldstein, Raymond E

    2012-09-18

    Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

  9. Factors that Control Mitotic Spindle Dynamics.

    PubMed

    Fraschini, Roberta

    2017-01-01

    Mitosis is the last phase of the cell cycle and it leads to the formation of two daughter cells with the same genetic information. This process must occurr in a very precise way and this task is essential to preserve genetic stability and to maintain cell viability. Accurate chromosome segregation during mitosis is brought about by an important cellular organelle: the mitotic spindle. This structure is made of microtubules, polymers of alpha and beta tubulin, and it is highly dynamic during the cell cycle: it emanates from two microtubules organizing centers (Spindle Pole Bodies, SPBs, in yeast) that are essential to build a short bipolar spindle, and it undergoes two steps of elongation during anaphase A and anaphase B in order to separate sister chromatids. Several proteins are involved in the control of mitotic spindle dynamics and their activity is tightly coordinated with other cell cycle events and with cell cycle progression.

  10. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  11. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  12. Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

    PubMed Central

    1995-01-01

    In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo. PMID:7490281

  13. Mechanical stability of bipolar spindle assembly

    NASA Astrophysics Data System (ADS)

    Malgaretti, Paolo; Muhuri, Sudipto

    2016-07-01

    Assembly and stability of mitotic spindle are governed by the interplay of various intra-cellular forces, e.g. the forces generated by motor proteins by sliding overlapping anti-parallel microtubules (MTs) polymerized from the opposite centrosomes, the interaction of kinetochores with MTs, and the interaction of MTs with the chromosome arms. We study the mechanical behavior and stability of spindle assembly within the framework of a minimal model which includes all these effects. For this model, we derive a closed-form analytical expression for the force acting between the centrosomes as a function of their separation distance and we show that an effective potential can be associated with the interactions at play. We obtain the stability diagram of spindle formation in terms of parameters characterizing the strength of motor sliding, repulsive forces generated by polymerizing MTs, and the forces arising out of the interaction of MTs with kinetochores. The stability diagram helps in quantifying the relative effects of the different interactions and elucidates the role of motor proteins in formation and inhibition of spindle structures during mitotic cell division. We also predict a regime of bistability for a certain parameter range, wherein the spindle structure can be stable for two different finite separation distances between centrosomes. This occurrence of bistability also suggests the mechanical versatility of such self-assembled spindle structures.

  14. Dissection of the NUP107 nuclear pore subcomplex reveals a novel interaction with spindle assembly checkpoint protein MAD1 in Caenorhabditis elegans

    PubMed Central

    Ródenas, Eduardo; González-Aguilera, Cristina; Ayuso, Cristina; Askjaer, Peter

    2012-01-01

    Nuclear pore complexes consist of several subcomplexes. The NUP107 complex is important for nucleocytoplasmic transport, nuclear envelope assembly, and kinetochore function. However, the underlying molecular mechanisms and the roles of individual complex members remain elusive. We report the first description of a genetic disruption of NUP107 in a metazoan. Caenorhabditis elegans NUP107/npp-5 mutants display temperature-dependent lethality. Surprisingly, NPP-5 is dispensable for incorporation of most nucleoporins into nuclear pores and for nuclear protein import. In contrast, NPP-5 is essential for proper kinetochore localization of NUP133/NPP-15, another NUP107 complex member, whereas recruitment of NUP96/NPP-10C and ELYS/MEL-28 is NPP-5 independent. We found that kinetochore protein NUF2/HIM-10 and Aurora B/AIR-2 kinase are less abundant on mitotic chromatin upon NPP-5 depletion. npp-5 mutants are hypersensitive to anoxia, suggesting that the spindle assembly checkpoint (SAC) is compromised. Indeed, NPP-5 interacts genetically and physically with SAC protein MAD1/MDF-1, whose nuclear envelope accumulation requires NPP-5. Thus our results strengthen the emerging connection between nuclear pore proteins and chromosome segregation. PMID:22238360

  15. Brownian dynamics simulation of fission yeast mitotic spindle formation

    NASA Astrophysics Data System (ADS)

    Edelmaier, Christopher

    2014-03-01

    The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies.

  16. A method for multiprotein assembly in cells reveals independent action of kinesins in complex

    PubMed Central

    Norris, Stephen R.; Soppina, Virupakshi; Dizaji, Aslan S.; Schimert, Kristin I.; Sept, David; Cai, Dawen; Sivaramakrishnan, Sivaraj

    2014-01-01

    Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track. PMID:25365993

  17. Fluctuation in the microtubule sliding movement driven by kinesin in vitro.

    PubMed

    Imafuku, Y; Toyoshima, Y Y; Tawada, K

    1996-02-01

    We studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calculating the mean-square displacement deviation from the average as a function of time, we obtained motional diffusion coefficients for microtubules and analyzed the dependence of the coefficients on microtubule length. Our analyses suggest that 1) the motional diffusion coefficient consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coefficient of a filament in Brownian movement is) and another that is independent of the length, and 2) the length-dependent term decreases with increasing kinesin concentration. This latter term almost vanishes within the length range we studied at high kinesin concentrations. From the length-dependence relationship, we evaluated the friction coefficient for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coefficient observed at sufficiently high kinesin concentrations indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.

  18. Properties of the Kinesin-1 motor DdKif3 from Dictyostelium discoideum.

    PubMed

    Röhlk, Christian; Rohlfs, Meino; Leier, Sven; Schliwa, Manfred; Liu, Xiao; Parsch, John; Woehlke, Günther

    2008-04-01

    The amoeba Dictyostelium discoideum possesses genes for 13 different kinesins. Here we characterize DdKif3, a member of the Kinesin-1 family. Kinesin-1 motors form homodimers that can move micrometer-long distances on microtubules using the energy derived from ATP hydrolysis. We expressed recombinant motors in Escherichia coli and tested them in different in vitro assays. Full-length and truncated Kif3 motors were active in gliding and ATPase assays. They showed a strong dependence on ionic strength. Like the full-length motor, the truncated DdKif3-592 motor (aa 1-592; comprising motor domain, neck, and partial stalk) reached its maximum speed of around 2.0micrcom s(-1) at a potassium acetate concentration of 200mM. The shortened DdKif3-342 motor (aa 1-342; comprising motor domain, partial neck) showed a high ATP turnover, comparable to that of the fungal Kinesin-1, Nkin. Results from the duty cycle calculations and gliding assays indicate that DdKif3 is a processive motor. A GFP-fusion protein revealed a mainly cytoplasmic localization of DdKif3. Immunofluorescence staining makes an association with the endoplasmic reticulum or mitochondria unlikely. Despite a similar phylogenetic distance to both metazoa and fungi, in terms of its biochemical properties DdKif3 revealed a closer similarity to fungal than animal kinesins.

  19. The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties.

    PubMed Central

    Steinberg, G; Schliwa, M

    1995-01-01

    The "conventional" kinesins comprise a conserved family of molecular motors for organelle transport that have been identified in various animal species. Organelle motors from other phyla have not yet been analyzed at the molecular level. Here we report the identification, biochemical and immunological characterization, and molecular cloning of a cytoplasmic motor in a "lower" eukaryote, the Ascomycete fungus Neurospora crassa. This motor, termed Nkin (for Neurospora kinesin), exhibits several unique structural and functional features, including a high rate of microtubule transport, a lack of copurifying light chains, a second P-loop motif, and an overall sequence organization reminiscent of a kinesin-like protein. However, a greater than average sequence homology in the motor domain and the presence of a highly conserved region in the C-terminus identify Nkin as a distant relative of the family of conventional kinesins. A molecular phylogenetic analysis suggests Nkin to have diverged early in the evolution of this family of motors. The discovery of Nkin may help identify domains important for specific biological functions in conventional kinesins. Images PMID:8589459

  20. BNIP-2 binds phosphatidylserine, localizes to vesicles, and is transported by kinesin-1.

    PubMed

    Akamatsu, Rie; Ishida-Kitagawa, Norihiro; Aoyama, Takane; Oka, Chio; Kawaichi, Masashi

    2015-02-01

    BNIP-2 shows high homology with the Cayman ataxia protein, caytaxin, which functions as a kinesin-1 adapter bridging cargos and kinesin light chains (KLCs). BNIP-2 is known to induce cell shape changes when over-expressed in culture cells, but its physiological functions are mostly unknown. BNIP-2 interacts with KLC through the conserved WED motif in the N-terminal region of BNIP-2. Interaction with KLC and transportation by kinesin-1 are essential for over-expressed BNIP-2 to elongate cells and induce cellular processes. Endogenous BNIP-2 localizes to the Golgi apparatus, early and recycling endosomes and mitochondria, aligned with microtubules, and moves at a speed compatible with kinesin-1 transportation. The CRAL-TRIO domain of BNIP-2 specifically interacts with phosphatidylserine, and the vesicular localization of BNIP-2 requires interaction with this phospholipid. BNIP-2 mutants which do not bind phosphatidylserine do not induce morphological changes in cells. These data show that similar to caytaxin, BNIP-2 is a kinesin-1 adapter involved in vesicular transportation in the cytoplasm and that association with cargos depends on interaction of the CRAL-TRIO domain with membrane phosphatidylserine.

  1. Expression of the mitotic motor protein Eg5 in postmitotic neurons: implications for neuronal development.

    PubMed

    Ferhat, L; Cook, C; Chauviere, M; Harper, M; Kress, M; Lyons, G E; Baas, P W

    1998-10-01

    It is well established that the microtubules of the mitotic spindle are organized by a variety of motor proteins, and it appears that the same motors or closely related variants organize microtubules in the postmitotic neuron. Specifically, cytoplasmic dynein and the kinesin-related motor known as CHO1/MKLP1 are used within the mitotic spindle, and recent studies suggest that they are also essential for the establishment of the axonal and dendritic microtubule arrays of the neuron. Other motors are required to tightly regulate microtubule behaviors in the mitotic spindle, and it is attractive to speculate that these motors might also help to regulate microtubule behaviors in the neuron. Here we show that a homolog of the mitotic kinesin-related motor known as Eg5 continues to be expressed in rodent neurons well after their terminal mitotic division. In neurons, Eg5 is directly associated with the microtubule array and is enriched within the distal regions of developing processes. This distal enrichment is transient, and typically lost after a process has been clearly defined as an axon or a dendrite. Strong expression can resume later in development, and if so, the protein concentrates within newly forming sprouts at the distal tips of dendrites. We suggest that Eg5 generates forces that help to regulate microtubule behaviors within the distal tips of developing axons and dendrites.

  2. Kinesin-2 family in vertebrate ciliogenesis

    PubMed Central

    Zhao, Chengtian; Omori, Yoshihiro; Brodowska, Katarzyna; Kovach, Peter; Malicki, Jarema

    2012-01-01

    The differentiation of cilia is mediated by kinesin-driven transport. As the function of kinesins in vertebrate ciliogenesis is poorly characterized, we decided to determine the role of kinesin-2 family motors—heterotrimeric kinesin-II and the homodimeric Kif17 kinesin—in zebrafish cilia. We report that kif17 is largely dispensable for ciliogenesis; kif17 homozygous mutant animals are viable and display subtle morphological defects of olfactory cilia only. In contrast to that, the kif3b gene, encoding a heterotrimeric kinesin subunit, is necessary for cilia differentiation in most tissues, although exceptions exist, and include photoreceptors and a subset of hair cells. Cilia of these cell types persist even in kif3b/kif17 double mutants. Although we have not observed a functional redundancy of kif3b and kif17, kif17 is able to substitute for kif3b in some cilia. In contrast to kif3b/kif17 double mutants, simultaneous interference with kif3b and kif3c leads to the complete loss of photoreceptor and hair cell cilia, revealing redundancy of function. This is in agreement with the idea that Kif3b and Kif3c motor subunits form complexes with Kif3a, but not with each other. Interestingly, kif3b mutant photoreceptor cilia differentiate with a delay, suggesting that kif3c, although redundant with kif3b at later stages of differentiation, is not active early in photoreceptor ciliogenesis. Consistent with that, the overexpression of kif3c in kif3b mutants rescues early photoreceptor cilia defects. These data reveal unexpected diversity of functional relationships between vertebrate ciliary kinesins, and show that the repertoire of kinesin motors changes in some cilia during their differentiation. PMID:22308397

  3. Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages.

    PubMed

    Charlebois, Blake D; Kollu, Swapna; Schek, Henry T; Compton, Duane A; Hunt, Alan J

    2011-04-06

    During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity-a mechanical paradigm that may be important throughout the spindle.

  4. Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes.

    PubMed

    Akendengue, Lurlène; Trépout, Sylvain; Graña, Martín; Voegele, Alexis; Janke, Carsten; Raynal, Bertrand; Chenal, Alexandre; Marco, Sergio; Wehenkel, Anne Marie

    2017-03-30

    Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today's eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.

  5. Kinesin-1 Translocation along Human Breast Cancer Cell Microtubules in Vitro

    NASA Astrophysics Data System (ADS)

    Shojania Feizabadi, Mitra; Jun, Yonggun

    2015-03-01

    A principle approach to better understand intra-cellular microtubule based transport is to study such it in vitro. Such in vitro examinations have predominantly used microtubules polymerized from bovine brain tubulin, but motor function can also in principle be affected by the specific tubulin isotypes present in different cells. The human breast cancer cells carry different beta tubulin isotype distribution. However, it is entirely unknown whether transport along the microtubules is different in these cells. In this work we have characterized, for the first time, the translocation specifications of kinesin-1 along human breast cancer cell microtubules polymerized in vitro. We found that as compared with the translocation along bovine brain microtubules, kinesin-1 shows a fifty percent shorter processive run length and slightly slower velocity under similar experimental conditions. These first time results support the regulatory role of tubulin isotypes in regards to motor protein translocations, and quantify the translocation specifications of kinesin-1 along microtubules of human breast cancer cells.

  6. Asymmetries in kinesin-2 and cytoplasmic dynein contributions to melanosome transport.

    PubMed

    De Rossi, María Cecilia; De Rossi, María Emilia; Sued, Mariela; Rodríguez, Daniela; Bruno, Luciana; Levi, Valeria

    2015-09-14

    The mechanisms involved in bidirectional transport along microtubules remain largely unknown. We explored the collective action of kinesin-2 and dynein motors during transport of melanosomes in Xenopus laevis melanophores. These motors are attached to organelles through accessory proteins establishing a complex molecular linker. We determined both the stiffness of this linker and the organelles speed and observed that these parameters depended on the organelle size and cargo direction. Our results suggest that melanosome transport is driven by two dissimilar teams: whereas dynein motors compete with kinesin-2 affecting the properties of plus-end directed organelles, kinesin-2 does not seem to play a similar role during minus-end transport. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Kinesin-1 plays a role in transport of SNAP-25 to the plasma membrane

    SciTech Connect

    Morton, April M.; Cunningham, Anthony L.; Diefenbach, Russell J.

    2010-01-01

    The cellular molecular motor kinesin-1 mediates the microtubule-dependent transport of a range of cargo. We have previously identified an interaction between the cargo-binding domain of kinesin-1 heavy chain KIF5B and the membrane-associated SNARE proteins SNAP-25 and SNAP-23. In this study we further defined the minimal SNAP-25 binding domain in KIF5B to residues 874-894. Overexpression of a fragment of KIF5B (residues 594-910) resulted in significant colocalization with SNAP-25 with resulting blockage of the trafficking of SNAP-25 to the periphery of cells. This indicates that kinesin-1 facilitates the transport of SNAP-25 containing vesicles as a prerequisite to SNAP-25 driven membrane fusion events.

  8. Microtubule shuttles on kinesin-coated glass micro-wire tracks.

    PubMed

    Kim, Kyongwan; Liao, Andrew L; Sikora, Aurélien; Oliveira, Daniel; Nakazawa, Hikaru; Umetsu, Mitsuo; Kumagai, Izumi; Adschiri, Tadafumi; Hwang, Wonmuk; Teizer, Winfried

    2014-08-01

    Gliding of microtubule filaments on surfaces coated with the motor protein kinesin has potential applications for nano-scale devices. The ability to guide the gliding direction in three dimensions allows the fabrication of tracks of arbitrary geometry in space. Here, we achieve this by using kinesin-coated glass wires of micrometer diameter range. Unlike previous methods in which the guiding tracks are fixed on flat two-dimensional surfaces, the flexibility of glass wires in shape and size facilitates building in-vitro devices that have deformable tracks.

  9. Profiling of the Mammalian Mitotic Spindle Proteome Reveals an ER Protein, OSTD-1, as Being Necessary for Cell Division and ER Morphology

    PubMed Central

    Bonner, Mary Kate; Han, Bo Hwa; Skop, Ahna

    2013-01-01

    Cell division is important for many cellular processes including cell growth, reproduction, wound healing and stem cell renewal. Failures in cell division can often lead to tumors and birth defects. To identify factors necessary for this process, we implemented a comparative profiling strategy of the published mitotic spindle proteome from our laboratory. Of the candidate mammalian proteins, we determined that 77% had orthologs in Caenorhabditis elegans and 18% were associated with human disease. Of the C. elegans candidates (n=146), we determined that 34 genes functioned in embryonic development and 56% of these were predicted to be membrane trafficking proteins. A secondary, visual screen to detect distinct defects in cell division revealed 21 genes that were necessary for cytokinesis. One of these candidates, OSTD-1, an ER resident protein, was further characterized due to the aberrant cleavage furrow placement and failures in division. We determined that OSTD-1 plays a role in maintaining the dynamic morphology of the ER during the cell cycle. In addition, 65% of all ostd-1 RNAi-treated embryos failed to correctly position cleavage furrows, suggesting that proper ER morphology plays a necessary function during animal cell division. PMID:24130834

  10. Aurora B prevents premature removal of spindle assembly checkpoint proteins from the kinetochore: A key role for Aurora B in mitosis.

    PubMed

    Gurden, Mark D; Anderhub, Simon J; Faisal, Amir; Linardopoulos, Spiros

    2016-07-18

    Accurate chromosome segregation is dependent on the spindle assembly checkpoint (SAC). In current models, the key direct role of Aurora B in the SAC has been suggested to be to promote rapid kinetochore localisation of MPS1, allowing MPS1 to generate the checkpoint signal. However, Aurora B is also thought to play an indirect role in the SAC through the destabilisation of kinetochore-microtubule (KT-MT) attachments. Here, we demonstrate that Aurora B activity is not required for the kinetochore recruitment of the majority of SAC proteins. More importantly, we show that the primary role of Aurora B in the SAC is to prevent the premature removal of SAC proteins from the kinetochore, which is strictly dependent on KT-MT interactions. Moreover, in the presence of KT-MT interactions, Aurora B inhibition silences a persistent SAC induced by tethering MPS1 to the kinetochore. This explains the highly synergistic interaction between Aurora B and MPS1 inhibitors to override the SAC, which is lost when cells are pre-arrested in nocodazole. Furthermore, we show that Aurora B and MPS1 inhibitors synergistically kill a panel of breast and colon cancer cell lines, including cells that are otherwise insensitive to Aurora B inhibitors alone. These data demonstrate that the major role of Aurora B in SAC is to prevent the removal of SAC proteins from tensionless kinetochores, thus inhibiting premature SAC silencing, and highlights a therapeutic strategy through combination of Aurora B and MPS1 inhibitors.

  11. Inscuteable Regulates the Pins-Mud Spindle Orientation Pathway

    PubMed Central

    Mauser, Jonathon F.; Prehoda, Kenneth E.

    2012-01-01

    During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud). PMID:22253744

  12. The chromokinesin Kid is required for maintenance of proper metaphase spindle size.

    PubMed

    Tokai-Nishizumi, Noriko; Ohsugi, Miho; Suzuki, Emiko; Yamamoto, Tadashi

    2005-11-01

    The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes-imprecise chromosome alignment at metaphase and shortened spindles- exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.

  13. Aurora A Phosphorylates MCAK to Control Ran-dependent Spindle Bipolarity

    PubMed Central

    Zhang, Xin; Ems-McClung, Stephanie C.

    2008-01-01

    During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition, we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity. PMID:18434591

  14. Aurora A phosphorylates MCAK to control ran-dependent spindle bipolarity.

    PubMed

    Zhang, Xin; Ems-McClung, Stephanie C; Walczak, Claire E

    2008-07-01

    During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition, we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity.

  15. Purification and partial characterization of the major "pathogenesis-related" tomato leaf protein P14 from potato spindle tuber viroid (PSTV)-infected tomato leaves.

    PubMed

    Camacho Henriquez, A; Sänger, H L

    1984-01-01

    The acid-extractable leaf proteins of potato spindle tuber viroid (PSTV) infected tomato plants were analysed electrophoretically on polyacrylamide gels. The most prominent alteration found during disease development was the appearance of a "pathogenesis-related" protein with an apparent molecular weight of 14,000 (called P14) which is drastically increased in concentration. Its induction, however, is not viroid-specific because it is also accumulating after viral and fungal infections. The degree of P14 accumulation could be directly correlated with the severity of the disease symptoms and its concentration was found to be highest in leaves of the tomato cultivar "Rutgers" four weeks after infection. P14 was isolated from such leaf material by acid-extraction of the leaf proteins, which were concentrated from the clarified homogenates by ultrafiltration through hollow fiber systems or by precipitation at 60 per cent ammonium sulphate saturation. P14 was finally purified by ion exchange chromatography on sulfopropyl (SP-C25) Sephadex and on DEAE cellulose. A protein with properties similar to those of P14 could also be isolated from healthy tomato leaves, where its concentration is about forty to fifty times lower than PSTV-infected tissue. P14 can be stained with Coomassie Brilliant Blue, silver and ethidium bromide, it is sensitive to digestion with pronase and not altered when treated with RNase and DNase. P14 is a basic protein with an estimated isoelectric point of 10.7 and its unusual behaviour during ultrafiltration indicates that it represents an elongated rather than a globular molecule in solution. P14 seems to be different from any of the so-called "pathogenesis-related" proteins described so far in Gynura aurantiaca, "Etrog" citron, potato and tomato after viroid-infection and in tobacco, cucumber and bean leaves after virus- or fungus-induced hypersensitive reactions.

  16. Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes

    PubMed Central

    Richardson, Dale N; Simmons, Mark P; Reddy, Anireddy SN

    2006-01-01

    Background Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved ~350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis) has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships. Results We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages. Conclusion The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage-specific groups contributed to the

  17. Computational fragment-based drug design to explore the hydrophobic sub-pocket of the mitotic kinesin Eg5 allosteric binding site

    NASA Astrophysics Data System (ADS)

    Oguievetskaia, Ksenia; Martin-Chanas, Laetitia; Vorotyntsev, Artem; Doppelt-Azeroual, Olivia; Brotel, Xavier; Adcock, Stewart A.; de Brevern, Alexandre G.; Delfaud, Francois; Moriaud, Fabrice

    2009-08-01

    Eg5, a mitotic kinesin exclusively involved in the formation and function of the mitotic spindle has attracted interest as an anticancer drug target. Eg5 is co-crystallized with several inhibitors bound to its allosteric binding pocket. Each of these occupies a pocket formed by loop 5/helix α2 (L5/α2). Recently designed inhibitors additionally occupy a hydrophobic pocket of this site. The goal of the present study was to explore this hydrophobic pocket with our MED-SuMo fragment-based protocol, and thus discover novel chemical structures that might bind as inhibitors. The MED-SuMo software is able to compare and superimpose similar interaction surfaces upon the whole protein data bank (PDB). In a fragment-based protocol, MED-SuMo retrieves MED-Portions that encode protein-fragment binding sites and are derived from cross-mining protein-ligand structures with libraries of small molecules. Furthermore we have excluded intra-family MED-Portions derived from Eg5 ligands that occupy the hydrophobic pocket and predicted new potential ligands by hybridization that would fill simultaneously both pockets. Some of the latter having original scaffolds and substituents in the hydrophobic pocket are identified in libraries of synthetically accessible molecules by the MED-Search software.

  18. Flipping a phosphate switch on kinesin-II to turn IFT around.

    PubMed

    Craige, Branch; Witman, George B

    2014-09-08

    Cilia and flagella are assembled and maintained by the motor-driven, bidirectional traffic of large protein complexes in a process termed intraflagellar transport (IFT). In this issue of Developmental Cell, Liang et al. (2014) report that IFT is regulated in part by the phosphorylation status of the kinesin-II subunit FLA8/KIF3B.

  19. Self-Organization and Forces in the Mitotic Spindle.

    PubMed

    Pavin, Nenad; Tolić, Iva M

    2016-07-05

    At the onset of division, the cell forms a spindle, a precise self-constructed micromachine composed of microtubules and the associated proteins, which divides the chromosomes between the two nascent daughter cells. The spindle arises from self-organization of microtubules and chromosomes, whose different types of motion help them explore the space and eventually approach and interact with each other. Once the interactions between the chromosomes and the microtubules have been established, the chromosomes are moved to the equatorial plane of the spindle and ultimately toward the opposite spindle poles. These transport processes rely on directed forces that are precisely regulated in space and time. In this review, we discuss how microtubule dynamics and their rotational movement drive spindle self-organization, as well as how the forces acting in the spindle are generated, balanced, and regulated.

  20. The spindle checkpoint and chromosome segregation in meiosis.

    PubMed

    Gorbsky, Gary J

    2015-07-01

    The spindle checkpoint is a key regulator of chromosome segregation in mitosis and meiosis. Its function is to prevent precocious anaphase onset before chromosomes have achieved bipolar attachment to the spindle. The spindle checkpoint comprises a complex set of signaling pathways that integrate microtubule dynamics, biomechanical forces at the kinetochores, and intricate regulation of protein interactions and post-translational modifications. Historically, many key observations that gave rise to the initial concepts of the spindle checkpoint were made in meiotic systems. In contrast with mitosis, the two distinct chromosome segregation events of meiosis present a special challenge for the regulation of checkpoint signaling. Preservation of fidelity in chromosome segregation in meiosis, controlled by the spindle checkpoint, also has a significant impact in human health. This review highlights the contributions from meiotic systems in understanding the spindle checkpoint as well as the role of checkpoint signaling in controlling the complex divisions of meiosis.

  1. Probing the mechanical architecture of the vertebrate meiotic spindle.

    PubMed

    Itabashi, Takeshi; Takagi, Jun; Shimamoto, Yuta; Onoe, Hiroaki; Kuwana, Kenta; Shimoyama, Isao; Gaetz, Jedidiah; Kapoor, Tarun M; Ishiwata, Shin'ichi

    2009-02-01

    Accurate chromosome segregation during meiosis depends on the assembly of a microtubule-based spindle of proper shape and size. Current models for spindle-size control focus on reaction diffusion-based chemical regulation and balance in activities of motor proteins. Although several molecular perturbations have been used to test these models, controlled mechanical perturbations have not been possible. Here we report a piezoresistive dual cantilever-based system to test models for spindle-size control and examine the mechanical features, such as deformability and stiffness, of the vertebrate meiotic spindle. We found that meiotic spindles prepared in Xenopus laevis egg extracts were viscoelastic and recovered their original shape in response to small compression. Larger compression resulted in plastic deformation, but the spindle adapted to this change, establishing a stable mechanical architecture at different sizes. The technique we describe here may also be useful for examining the micromechanics of other cellular organelles.

  2. Initial conformation of kinesin's neck linker

    NASA Astrophysics Data System (ADS)

    Geng, Yi-Zhao; Ji, Qing; Liu, Shu-Xia; Yan, Shi-Wei

    2014-10-01

    How ATP binding initiates the docking process of kinesin's neck linker is a key question in understanding kinesin mechanisms. By exploiting a molecular dynamics method, we investigate the initial conformation of kinesin's neck linker in its docking process. We find that, in the initial conformation, the neck linker has interactions with β0 and forms a ‘cover-neck bundle’ structure with β0. From this initial structure, the formation of extra turns and the docking of the cover-neck bundle structure can be achieved. The motor head provides a forward force on the initial cover-neck bundle structure through ATP-induced rotation. This force, together with the hydrophobic interaction of ILE327 with the hydrophobic pocket on the motor head, drives the formation of the extra turn and initiates the neck linker docking process. Based on these findings, a pathway from ATP binding-induced motor head rotation to neck linker docking is proposed.

  3. Moonlighting Motors: Kinesin, Dynein, and Cell Polarity.

    PubMed

    Lu, Wen; Gelfand, Vladimir I

    2017-03-08

    In addition to their well-known role in transporting cargoes in the cytoplasm, microtubule motors organize their own tracks - the microtubules. While this function is mostly studied in the context of cell division, it is essential for microtubule organization and generation of cell polarity in interphase cells. Kinesin-1, the most abundant microtubule motor, plays a role in the initial formation of neurites. This review describes the mechanism of kinesin-1-driven microtubule sliding and discusses its biological significance in neurons. Recent studies describing the interplay between kinesin-1 and cytoplasmic dynein in the translocation of microtubules are discussed. In addition, we evaluate recent work exploring the developmental regulation of microtubule sliding during axonal outgrowth and regeneration. Collectively, the discussed works suggest that sliding of interphase microtubules by motors is a novel force-generating mechanism that reorganizes the cytoskeleton and drives shape change and polarization.

  4. Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans

    PubMed Central

    Burghoorn, Jan; Dekkers, Martijn P. J.; Rademakers, Suzanne; de Jong, Ton; Willemsen, Rob; Jansen, Gert

    2007-01-01

    In the cilia of the nematode Caenorhabditis elegans, anterograde intraflagellar transport (IFT) is mediated by two kinesin-2 complexes, kinesin II and OSM-3 kinesin. These complexes function together in the cilia middle segments, whereas OSM-3 alone mediates transport in the distal segments. Not much is known about the mechanisms that compartmentalize the kinesin-2 complexes or how transport by both kinesins is coordinated. Here, we identify DYF-5, a conserved MAP kinase that plays a role in these processes. Fluorescence microscopy and EM revealed that the cilia of dyf-5 loss-of-function (lf) animals are elongated and are not properly aligned into the amphid channel. Some cilia do enter the amphid channel, but the distal ends of these cilia show accumulation of proteins. Consistent with these observations, we found that six IFT proteins accumulate in the cilia of dyf-5(lf) mutants. In addition, using genetic analyses and live imaging to measure the motility of IFT proteins, we show that dyf-5 is required to restrict kinesin II to the cilia middle segments. Finally, we show that, in dyf-5(lf) mutants, OSM-3 moves at a reduced speed and is not attached to IFT particles. We propose that DYF-5 plays a role in the undocking of kinesin II from IFT particles and in the docking of OSM-3 onto IFT particles. PMID:17420466

  5. Balanced activity of three mitotic motors is required for bipolar spindle assembly and chromosome segregation.

    PubMed

    van Heesbeen, Roy G H P; Tanenbaum, Marvin E; Medema, René H

    2014-08-21

    Bipolar spindle assembly requires force to organize the microtubule network. Here, we show that three motor proteins, namely Eg5, Kif15, and dynein, act together to produce the right force balance in the spindle. Excessive inward force results in monopolar spindle formation, while excessive outward force generation results in unstable spindles with splayed spindle poles. Blocking activity of all three motors prevents bipolar spindle formation, but established bipolar spindles are refractory to loss of all motor activity. Further analysis shows that although these preformed spindles remain bipolar, outward force generation is required to establish sufficient tension on kinetochores and to accomplish successful chromosome segregation. Together, these results show how Eg5, Kif15, and dynein work together to build a bipolar spindle and reveal an important role for antagonistic motors in chromosome segregation.

  6. Myosin-10 independently influences mitotic spindle structure and mitotic progression.

    PubMed

    Sandquist, Joshua C; Larson, Matthew E; Hine, Ken J

    2016-06-01

    The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein's spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10's close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10's spindle functions require microtubule binding. In particular, myosin-10's contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. © 2016 Wiley Periodicals, Inc.

  7. Solution structure of two molecular motor domains: nonclaret disjunctional and kinesin.

    PubMed Central

    Eden, D; Luu, B Q; Zapata, D J; Sablin, E P; Kull, F J

    1995-01-01

    The effects of selected ligands on the structure of the truncated heavy-chain chemomechanical motor domains of Drosophila ncd and human kinesin were compared using the technique of transient electric birefringence. The 366-amino acid C-terminal motor domain of Drosophila nonclaret disjunctional, ncd(335-700), and the 349-amino acid N-terminal motor domain of human kinesin, kinesin(349), were studied at 4 degrees C in neutral buffers with ionic strength of 100 mM to form complexes with either MgADP or MgADP.Vi. The rotational diffusion time adjusted to 20 degrees C and water, tau 20,W, for ncd(335-700).MgADP is 32.8 ns, and for ncd(335-700).MgADP.Vi is 34.8 ns, suggesting prolate ellipsoids with dimensions 9.40 x 3.77 nm and 9.73 x 3.70 nm, respectively. The specific Kerr constant, Ksp, of ncd is -1.65 x 10(-12) cm2V-2 for the MgADP complex and -1.15 x 10(-12) cm2V-2 for the MgADP.Vi complex. The large negative Ksp for a prolate protein suggests an unusual charge distribution with two long surfaces with opposite charge. The tau 20,W for kinesin(349).MgADP is longer than the corresponding ncd motor and shows a decrease with increased electric field. The kinesin(349).MgADP.Vi complex has a longer tau 20,W. The Ksp for kinesin(349) is 0.36 x 10(-12) cm2V-2 for each complex. PMID:7787102

  8. Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

    PubMed Central

    Schneider, René; Korten, Till; Walter, Wilhelm J.; Diez, Stefan

    2015-01-01

    Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimer’s. However, little is known about how motor proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motor’s specific intracellular tasks and environments. PMID:25954882

  9. Kinesin-1 motors can circumvent permanent roadblocks by side-shifting to neighboring protofilaments.

    PubMed

    Schneider, René; Korten, Till; Walter, Wilhelm J; Diez, Stefan

    2015-05-05

    Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimer's. However, little is known about how motor proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼ 0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼ 70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motor's specific intracellular tasks and environments.

  10. Proadrenomedullin N-terminal 20 peptide increases kinesin's velocity both in vitro and in vivo.

    PubMed

    Larráyoz, Ignacio M; Martínez, Alfredo

    2012-04-01

    Intracellular cargo transport relies on microtubules and motor proteins such as kinesins and dyneins. Currently we have ample knowledge of the mechanisms by which motor proteins propel themselves along the microtubules, but little is known about intracellular factors that regulate motor speed. Here we show that proadrenomedullin N-terminal 20 peptide (PAMP) increases kinesin velocity and ATP consumption in a dose-dependent manner, using a variety of human kinesins. Structure-activity studies found that the terminal amide of PAMP is required for modulating kinesin activity and that the smallest peptide fragment retaining this role is PAMP₁₂₋₂₀. On the other hand, peptide fragments as small as PAMP₁₈₋₂₀ maintained the ability of delaying tubulin polymerization, another function previously described for PAMP, indicating that these two activities depend on different regions of the molecule. To demonstrate that these observations are also relevant in vivo, hippocampal neurons were isolated from mice lacking the gene coding for PAMP and from wild type littermates. Intravital stains followed by time-lapse microscopy analysis revealed that mitochondrial speed inside neurons lacking PAMP was significantly slower than in cells expressing the peptide. External addition of synthetic PAMP reversed this phenotype in PAMP-null neurons. Besides the obvious implications for better understanding cell biology, these results may be also relevant for the rapidly evolving discipline of nanotechnology because PAMP may be used as an accelerator of nanodevices based on microtubules and motor proteins.

  11. Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility

    PubMed Central

    Sun, Faneng; Zhu, Chuanmei; Dixit, Ram; Cavalli, Valeria

    2011-01-01

    Neuronal development, function and repair critically depend on axonal transport of vesicles and protein complexes, which is mediated in part by the molecular motor kinesin-1. Adaptor proteins recruit kinesin-1 to vesicles via direct association with kinesin heavy chain (KHC), the force-generating component, or via the accessory light chain (KLC). Binding of adaptors to the motor is believed to engage the motor for microtubule-based transport. We report that the adaptor protein Sunday Driver (syd, also known as JIP3 or JSAP1) interacts directly with KHC, in addition to and independently of its known interaction with KLC. Using an in vitro motility assay, we show that syd activates KHC for transport and enhances its motility, increasing both KHC velocity and run length. syd binding to KHC is functional in neurons, as syd mutants that bind KHC but not KLC are transported to axons and dendrites similarly to wild-type syd. This transport does not rely on syd oligomerization with itself or other JIP family members. These results establish syd as a positive regulator of kinesin activity and motility. PMID:21750526

  12. Recycling of kinesin-1 motors by diffusion after transport.

    PubMed

    Blasius, T Lynne; Reed, Nathan; Slepchenko, Boris M; Verhey, Kristen J

    2013-01-01

    Kinesin motors drive the long-distance anterograde transport of cellular components along microtubule tracks. Kinesin-dependent transport plays a critical role in neurogenesis and neuronal function due to the large distance separating the soma and nerve terminal. The fate of kinesin motors after delivery of their cargoes is unknown but has been postulated to involve degradation at the nerve terminal, recycling via retrograde motors, and/or recycling via diffusion. We set out to test these models concerning the fate of kinesin-1 motors after completion of transport in neuronal cells. We find that kinesin-1 motors are neither degraded nor returned by retrograde motors. By combining mathematical modeling and experimental analysis, we propose a model in which the distribution and recycling of kinesin-1 motors fits a "loose bucket brigade" where individual motors alter between periods of active transport and free diffusion within neuronal processes. These results suggest that individual kinesin-1 motors are utilized for multiple rounds of transport.

  13. Allostery Wiring Map for Kinesin Energy Transduction and Its Evolution*

    PubMed Central

    Richard, Jessica; Kim, Elizabeth D.; Nguyen, Hoang; Kim, Catherine D.; Kim, Sunyoung

    2016-01-01

    How signals between the kinesin active and cytoskeletal binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and thus are predicted to form a complex, non-linear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop 5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily. PMID:27507814

  14. Specific kinesin expression profiles associated with taxane resistance in basal-like breast cancer.

    PubMed

    Tan, Min Han; De, Sarmishtha; Bebek, Gurkan; Orloff, Mohammed S; Wesolowski, Robert; Downs-Kelly, Erinn; Budd, G Thomas; Stark, George R; Eng, Charis

    2012-02-01

    Breast cancer is a genetically heterogenous disease with subtypes differing in prognosis and chemosensitivity. The basal-like breast cancer (BLBC) molecular subtype is associated with poorer outcomes, but is more responsive to taxane-based chemotherapy. Kinesins are intracellular transport proteins that interact with microtubules, which are also the mechanistic target for taxanes. We investigated the relationship between taxane resistance in BLBC and kinesins using both expression and functional studies. Kinesin (KIF) expression was evaluated in three settings in relation to taxane resistance: (i) the NCI-60 cancer cell lines, (ii) pre-treatment samples from four BLBC patient cohorts receiving neoadjuvant chemotherapy regimens with and without taxanes, and (iii) post-treatment samples from residual breast cancer following neoadjuvant taxane-containing chemotherapy. We used a novel functional approach to gene modification, validation-based insertional mutagenesis, to select kinesin-overexpressing clones of BLBC cells for evaluation of related mechanisms of taxane resistance. In the NCI-60 cell line dataset, overexpression of the kinesin KIFC3 is significantly correlated with resistance to both docetaxel (P < 0.001) and paclitaxel (P < 0.001), but not to platinum-based chemotherapy, including carboplatin (P = 0.49) and cisplatin (P = 0.10). Overexpression of KIFC3 and KIF5A in pre-chemotherapy samples similarly predicted resistance to paclitaxel in the MDACC cohorts (P = 0.01); no KIF predicted resistance to fluorouracil-epirubicin-cyclophosphamide or cisplatin in BLBC patient cohorts treated without taxanes. KIF12 is the most overexpressed KIF gene in post-chemotherapy taxane-resistant residual breast cancers (2.8-fold-change). Functional studies established that overexpression of KIFC3, KIF5A, and KIF12 were specific in mediating resistance to docetaxel and not vincristine or doxorubicin. Mutation of the ATP-binding domain of a kinesin abolished its

  15. Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domain

    PubMed Central

    Shapira, Ofer; Gheber, Larisa

    2016-01-01

    The Saccharomyces cerevisiae kinesin-5 Cin8 performs essential mitotic functions in spindle assembly and anaphase B spindle elongation. Recent work has shown that Cin8 is a bi-directional motor which moves towards the minus-end of microtubules (MTs) under high ionic strength (IS) conditions and changes directionality in low IS conditions and when bound between anti-parallel microtubules. Previous work from our laboratory has also indicated that Cin8 is differentially phosphorylated during late anaphase at cyclin-dependent kinase 1 (Cdk1)-specific sites located in its motor domain. In vivo, such phosphorylation causes Cin8 detachment from spindles and reduces the spindle elongation rate, while maintaining proper spindle morphology. To study the effect of phosphorylation on Cin8 motor function, we examined in vitro motile properties of wild type Cin8, as well as its phosphorylation using phospho-deficient and phospho-mimic variants, in a single molecule fluorescence motility assay. Analysis was performed on whole cell extracts and on purified Cin8 samples. We found that addition of negative charges in the phospho-mimic mutant weakened the MT-motor interaction, increased motor velocity and promoted minus-end-directed motility. These results indicate that phosphorylation in the catalytic domain of Cin8 regulates its motor function. PMID:27216310

  16. Induction of Aneuploidy, Centrosome Abnormality, Multipolar Spindle, and Multipolar Division in Cultured Mammalian Cells Exposed to an Arsenic Metabolite, Dimethylarsinate.

    PubMed

    Ochi, Takafumi

    2016-01-01

    Toxicological studies of arsenic compounds were conducted in cultured mammalian cells to investigate the effects of glutathione (GSH) depletion. Dimethylarsinate DMA(V) was not cytotoxic in cells depleted of GSH, but was found to be cytotoxic when GSH was present outside the cells. The results suggested that a reactive form of DMA(V) was generated through interaction with GSH. Dimethylarsine iodide DMI(III) was used as a model compound of DMA(III), and the biological effects were investigated. DMI(III) was about 10000 times more toxic to the cells than DMA(V). Chromosome structural aberrations and numerical changes, such as aneuploidy, were induced by DMI(III). DMA(V) induced multiple foci of the centrosome protein, γ-tubulin, which were colocalized with multipolar spindles in mitotic cells. The multiple foci coalesced into a single dot on disruption of the microtubules (MT). However, reorganization of the MT caused multiple foci of γ-tubulin, suggesting that the induction of centrosome abnormalities by DMA(V) required intact MT. Inhibition of the MT-dependent motor, kinesin, prevented formation of multiple foci of γ-tubulin, which pointed to the involvement of the MT-dependent mitotic motor, kinesin, in the maintenance of centrosome abnormalities. DMI(III) caused abnormal cytokinesis (multipolar division). In addition, DMI(III) caused morphological transformation in Syrian hamster embryo cells. Consideration of the overall process following the centrosome abnormalities caused by DMA(V) suggested a mode of cytotoxicity in which the mitotic centrosome is a critical target.

  17. Myosin-10 independently influences mitotic spindle structure and mitotic progression

    PubMed Central

    Sandquist, Joshua C.; Larson, Matthew E.; Hine, Ken J.

    2016-01-01

    The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein’s spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10’s close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10’s spindle functions require microtubule binding. In particular, myosin-10’s contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. PMID:27220038

  18. Inhibition of kinesin-5 improves regeneration of injured axons by a novel microtubule-based mechanism

    PubMed Central

    Baas, Peter W.; Matamoros, Andrew J.

    2015-01-01

    Microtubules have been identified as a powerful target for augmenting regeneration of injured adult axons in the central nervous system. Drugs that stabilize microtubules have shown some promise, but there are concerns that abnormally stabilizing microtubules may have only limited benefits for regeneration, while at the same time may be detrimental to the normal work that microtubules perform for the axon. Kinesin-5 (also called kif11 or Eg5), a molecular motor protein best known for its crucial role in mitosis, acts as a brake on microtubule movements by other motor proteins in the axon. Drugs that inhibit kinesin-5, originally developed to treat cancer, result in greater mobility of microtubules in the axon and an overall shift in the forces on the microtubule array. As a result, the axon grows faster, retracts less, and more readily enters environments that are inhibitory to axonal regeneration. Thus, drugs that inhibit kinesin-5 offer a novel microtubule-based means to boost axonal regeneration without the concerns that accompany abnormal stabilization of the microtubule array. Even so, inhibiting kinesin-5 is not without its own caveats, such as potential problems with navigation of the regenerating axon to its target, as well as morphological effects on dendrites that could affect learning and memory if the drugs reach the brain. PMID:26199587

  19. FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination.

    PubMed

    Yasuda, Kyota; Clatterbuck-Soper, Sarah F; Jackrel, Meredith E; Shorter, James; Mili, Stavroula

    2017-04-03

    Cytoplasmic inclusions of the RNA-binding protein fused in sarcoma (FUS) represent one type of membraneless ribonucleoprotein compartment. Formation of FUS inclusions is promoted by amyotrophic lateral sclerosis (ALS)-linked mutations, but the cellular functions affected upon inclusion formation are poorly defined. In this study, we find that FUS inclusions lead to the mislocalization of specific RNAs from fibroblast cell protrusions and neuronal axons. This is mediated by recruitment of kinesin-1 mRNA and protein within FUS inclusions, leading to a loss of detyrosinated glutamate (Glu)-microtubules (MTs; Glu-MTs) and an inability to support the localization of RNAs at protrusions. Importantly, dissolution of FUS inclusions using engineered Hsp104 disaggregases, or overexpression of kinesin-1, reverses these effects. We further provide evidence that kinesin-1 affects MT detyrosination not through changes in MT stability, but rather through targeting the tubulin carboxypeptidase enzyme onto specific MTs. Interestingly, other pathological inclusions lead to similar outcomes, but through apparently distinct mechanisms. These results reveal a novel kinesin-dependent mechanism controlling the MT cytoskeleton and identify loss of Glu-MTs and RNA mislocalization as common outcomes of ALS pathogenic mutations.

  20. Effects of potential environmental interferents on kinesin-powered molecular shuttles

    NASA Astrophysics Data System (ADS)

    Bachand, Marlene; Bachand, George D.

    2012-05-01

    Biomolecular motor-powered active transport represents an alternate means for analyte processing in nanoscale biosensors and bioanalytical devices. For example, a prototype ``smart dust'' biosensor has recently been reported in which the motor protein kinesin processes antibody-functionalized microtubules (MTs) to capture and separate optically tagged protein analytes. A potential limitation of this technology, however, involves the inhibition of transport function by interfering compounds that may be present in raw samples. Here we characterized the response of kinesin-MT transport to a range of potential interferents including solvents, acids, oxidizers, and environmental contaminants. The results of kinesin motility assays suggest that, among the tested interferents, only acetic acid and sodium hypochlorite adversely affected MT transport, primarily due to depolymerization of MT filaments. While negative effects were not observed for the remaining compounds tested, enhancement in motility was observed in the presence of acetone, antifreeze, and organic matter. Overall, the data suggest that kinesin-MT transport is resilient against a variety of common interferents, but primarily susceptible to failure due to significant changes in pH or the presence of an oxidizer.

  1. Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways.

    PubMed Central

    Geiser, J R; Schott, E J; Kingsbury, T J; Cole, N B; Totis, L J; Bhattacharyya, G; He, L; Hoyt, M A

    1997-01-01

    Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability. We identified 20 genes whose products are specifically required by cell deficient for Cin8p. All are associated with mitotic roles and represent at least four different functional pathways. These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway. In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered. Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect. We provide evidence that the products of these genes function in concept with dynein. Among the dynein pathway gene products, we found homologues of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development. These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family. Images PMID:9201714

  2. Cell Division: NuMA Bears the Load in the Spindle.

    PubMed

    Maiato, Helder; Pereira, António J

    2017-08-07

    The mitotic spindle bears the load of chromosomes during mitosis, but how this load is distributed across the spindle is unclear. A new study shows that load distribution in the spindle is confined and requires the microtubule cross-linking protein NuMA. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

    PubMed Central

    Colombié, Nathalie; Głuszek, A. Agata; Meireles, Ana M.; Ohkura, Hiroyuki

    2013-01-01

    In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which “amplifies” spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle. PMID:23785300

  4. Meiosis-specific stable binding of augmin to acentrosomal spindle poles promotes biased microtubule assembly in oocytes.

    PubMed

    Colombié, Nathalie; Głuszek, A Agata; Meireles, Ana M; Ohkura, Hiroyuki

    2013-06-01

    In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.

  5. The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly.

    PubMed

    Labbé, Jean-Claude; McCarthy, Erin K; Goldstein, Bob

    2004-10-25

    Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.

  6. Anthrax receptors position the spindle.

    PubMed

    Minc, Nicolas; Piel, Matthieu

    2013-01-01

    Spindle orientation plays a pivotal role in tissue morphogenesis. An asymmetric anthrax receptor cap is revealed to promote activation of a formin to orient the spindle along the planar cell polarity (PCP) axis in zebrafish dorsal epiblast cells.

  7. Kinesin-1/Hsc70-dependent mechanism of slow axonal transport and its relation to fast axonal transport

    PubMed Central

    Terada, Sumio; Kinjo, Masataka; Aihara, Makoto; Takei, Yosuke; Hirokawa, Nobutaka

    2010-01-01

    Cytoplasmic protein transport in axons (‘slow axonal transport') is essential for neuronal homeostasis, and involves Kinesin-1, the same motor for membranous organelle transport (‘fast axonal transport'). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin-1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ-like domain of the kinesin light chain in the Kinesin-1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin-1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant-negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction. PMID:20111006

  8. Diffusive Movement of Processive Kinesin-1 on Microtubules

    PubMed Central

    Lu, Hailong; Ali, M. Yusuf; Bookwalter, Carol S.; Warshaw, David M.; Trybus, Kathleen M.

    2009-01-01

    The processive motor kinesin-1 moves unidirectionally toward the plus end of microtubules. This process can be visualized by total internal reflection fluorescence (TIRF) microscopy of kinesin bound to a carboxylated Quantum dot (Qdot), which acts both as cargo and label. Surprisingly, when kinesin is bound to an anti-HIS Qdot, it shows diffusive movement on microtubules, which decreased in favor of processive runs with increasing salt concentration. This observation implies that kinesin movement on microtubules is governed by its conformation, as it is well-established that kinesin undergoes a salt-dependent transition from a folded (inactive) to an extended (active) molecule. A truncated kinesin lacking the last 75 amino acids (kinesin-ΔC) showed both processive and diffusive movement on microtubules. The extent of each behavior depends on the relative amounts of ADP and ATP, with purely diffusive movement occurring in ADP alone. Taken together, these data imply that folded kinesin.ADP can exist in a state that diffuses along the microtubule lattice without expending energy. This mechanism may facilitate the ability of kinesin to pick up cargo, and/or allow the kinesin/cargo complex to stay bound after encountering obstacles. PMID:19682327

  9. Evidence that the stalk of Drosophila kinesin heavy chain is an alpha- helical coiled coil

    PubMed Central

    1992-01-01

    Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40- 55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross- linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha- helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha- helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha- helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest t