Abstracts of Current Research:- The switchable directionality of the mitotic kinesin-5 motors: Activity of the conserved kinesin-5 motor proteins is among the major factors that govern the morphological changes of the mitotic spindle during cell division. Kinesin-5 motors function as homotetrames, with two pairs of catalytic domains located at the opposite sides of the elongated active complex. By this special architecture, kinesin-5 motors are believed to crosslink antiparallel spindle microtubules (MTs) and provide the spindle-pole separating force required for their mitotic functions in spindle assembly, maintenance of the bipolar spindle structure, and anaphase B spindle elongation. Although kinesin-5’s function is essential for mitosis throughout the eukaryotes, the mechanism and regulation of their functions remain to be elucidated. Here we propose to study the function and regulation of Cin8 and Kip1, the two kinesin-5 homologs of the genetically tractable eukaryote Saccharomyces cerevisiae.
The majority of the members of the kinesin superfamily are N-terminal, i.e., their catalytic domain is positioned at the N-terminus of the protein, and plus-end directed, i.e., they move towards the dynamic plusends of the MTs. Minus-end motility was seen only for the structurally distinct C-terminal kinesin-14 family members. Recently, we and another group reported that the N-terminal S. cerevisiae kinesin-5 Cin8 moves processively towards the minus-end of MTs and is able to switch directionality under certain experimental conditions (Gerson-Gurwitz et al., 2011; Roostalu et al., 2011). These findings break the long-standing dogma regarding plus-end directionality of N-terminal kinesins. However, basic questions remain unclear: what is the mechanism of the minus-end directed and switchable motility, what is its physiological importance and function, and how is the directionality of kinesin-5 motors regulated?
One of the goals of our research is to understand this challenging problem.
- Activity and regulation of HsEg5, a human mitotic kinesin-related protein.: The aim of the present project is to study the activity and regulation of HsEg5, a human KRP from the BimC family. In human cells, elimination of the HsEg5 function leads to accumulation of cells blocked in mitosis with abnormal spindles. Due to the absence of appropriate assays, HsEg5 motor activity in different stages of mitosis has never been studied nor has cell-cycle regulation of its motor activity been addresses. To study cell-cycle regulation of HsEg5 activity, we will develop a set of in vitro assays to analyze its motile properties directly from human cells, and not from an exogenous expression system. This will enable us to compare HsEg5 motor activity at specific points during the cell-cycle. Similar approach was recently developed by us for the study of motile properties of Cin8p, a BimC family member from the budding yeast Saccharomyces cerevisiae. To develop in vitro assays for HsEg5, we will subclone a biotin-tagging sequence at the end of the HsEg5 encoding sequence1 and will express the fusion protein in HT-29 human cell lines. We will analyze HsEg5-induced microtubule binding and motility from HT-29 cell extracts on streptavidin coated surface, using a video-enhanced Nomarski microscope to visualize single microtubules. Since HsEg5 motor activity in human cells was not studied before, as the first stage of this study, we will characterize the nucleotide specificity of HsEg5-induced microtubule binding, velocity of microtubule gliding and processivity, in HT-29 cells. To address cell-cycle regulation of HsEg5 activity, we will analyze its motile properties in extracts obtained from different cell-cycle points, including during mitosis. We will also address the role of phosphorylation in HsEg5 activity regulation in these extracts. To understand how HsEg5 motor activity is related to its mitotic roles, we will examine its expression and localization at the same cell-cycle points during which its motor activity is examined.It is our beli
- Regulation of Cin8p activity and its multiple mitotic roles: Evidence from genetic studies indicate that molecular motors from the BimC family of kinesin-related proteins, which have been found in numerous eukaryotic species, play central roles in mediating spindle dynamics. The aim of the present project is to study the activity and regulation of Cin8p, a Saccharomyces cerevisiae BimC family member. Although Cin8p activity was implicated in all stages of mitosis, very little is known about the mechanisms by which Cin8p performs its multiple mitotic roles and how its activity is regulated. To address this problem we propose to combine biophysical, cell-biology and genetic approaches to study cell-cycle regulation of Cin8p activity. We will apply our recently developed in vitro assays, which analyze Cin8p motile properties directly from S. cerevisiae cells1. Preliminary data obtained with these assays demonstrated for the first time that Cin8p motor activity changes during mitosis.To further characterize and understand this phenomenon we will study Cin8p-induced microtubule binding and motility in different points of the cell-cycle using a video-enhanced Nomarski microscope to visualize single microtubules. We will examine Cin8p phosphorylation throughout the cell-cycle and design Cin8p mutants defective for phosphorylation. Using these mutants we will determine in vivo roles for Cin8p phosphorylation, identify possible modulators of Cin8p phosphorylation stage and study the role of phosphorylation in modulating Cin8p motor activity.It is our belief that the suggested study will not only enlarge our understanding of the mechanisms of Cin8p activity regulation in S. cerevisiae, but will also shed light on the way BimC family members perform their mitotic roles.
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Publications:- C. Thiede, V. Fridman, A. Gerson-Gurwitz, L. Gheber, and C. F. Schmidt . Regulation of bi-directional movement of single kinesin-5 Cin8 molecules BioArchitecture 2: 70-74 (2012)
- A. Sadeh, N. Movshovich, M. Volokh, L. Gheber, A. Aharoni . Fine-Tuning of the Msn2/4-Mediated yeast stress responses as revealed by systematic deletion of Msn2/4 partners Molecular Biology of the Cell 22: 3127-38 (2011)
- R. Avunie-Masala, N. Movshovich, Y. Nissenkorn, A. Gerson-Gurwitz, V. Fridman, M. Kõivomägi, M. Loog, M.A. Hoyt, A. Zaritsky, L. Gheber. Phospho-regulation of Kinesin-5 function during anaphase spindle elongatio Journal of Cell Science 124: 873-8 (2011)
- A. Gerson-Gurwitz, C. Thiede, N. Movshovich, V. Fridman, M. Podolskaya, T. Danieli, S. Lakämper, D.R. Klopfenstein, C.F. Schmidt and L. Gheber. Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry EMBO Journal 30: 4942-54 (2011)
- A. Avraham, J. Sandbank, N. Yarom, A. Shalom, T. Karni, I. Pappo, A. Sella, A. Fich, S. Walfisch, L. Gheber, E. Evron. A similar cell specific pattern of HoxA methylation in normal and in cancer tissues Epigenetics 5(1): 1-6 (2010)
- O. Voloshin, Y. Gocheva, M. Gutnick, N. Movshovich, A. Bakhrat, K. Baranes-Bachar, D. Bar-Zvi, R. Parvari, L. Gheber, D. Raveh. Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress Cellular and Molecular Life Sciences 67: 2025-38 (2010)
- V. Fridman, A. Gerson-Gurwitz, N. Movshovich, M. Kupiec, L. Gheber. Midzone organization restricts interpolar microtubule plus-end dynamics during spindle elongation EMBO reports 10: 387-93 (2009)
- A. Gerson-Gurwitz, N. Movshovich, K. Moyal, R. Avunie-Masala, V. Fridman, MA. Hoyt, B. Katz, L. Gheber. Mid-anaphase arrest in cells eliminated for the function of Cin8 and dynein Cellular and Molecular Life Sciences 66: 301-13 (2009)
- N. Movshovich, V. Fridman, A. Gerson-Gurwitz, I. Schumacher, I. Gertsberg, A. Fich, MA. Hoyt, B. Katz, L. Gheber. Slk19-dependent mid-anaphase pause in kinesin-5 mutated cells Journal of Cell Science 121: 2529-39 (2008)
- N. Dubi, L. Gheber, D. Fishman, M Hershfinkel, and I. Sekler. Extracellular Zinc and Zink-citrate acting through a putative Zinc sensing receptor, regulate growth and survival of prostate cancer cell Carcinogenesis 29: 1692-700 (2008)
- E.R. Hildebrandt, L. Gheber, T. Kingsbury and M.A. Hoyt . Homotetrameric form of Cin8p, an S. cerevisiae kinesin-5 motor, is essential for its in vivo function Journal of Biological Chemistry 281: 26004-26013 (2006)
- A. Mermelshtein, A. Gerson, S. Walfisch, B. Delgado, G. Shechter-Maor, J. Delgado, A. Fich and L. Gheber.. Expression of D-type cyclins in colon cancer and in cell-lines from colon carcinomas British Journal of Cancer 93: 338-45 (2005)
- I. Leizerman, R. Avunie-Masala, M. Elkabets, A. Fich and L. Gheber . Differential effect of monastrol in two human cell lines Cellular and Molecular Life Sciences 61: 2060-2070 (2004)
- F. R. Cottingham, L. Gheber, D. L. Miller and M. Andrew Hoyt. Novel Roles for Saccharomyces cerevisiae Mitotic Spindle Motors The Journal of Cell Biology 147: 335-349 (2000)
- L. Gheber, S.C. Kuo and M.A. Hoyt. Motile properties of the kinesin-related Cin8p spindle motor extracted from S. cerevisiae cells Journal of Biological Chemistry 274: 9564-9572 (1999)
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