Publications by authors named "Larisa Gheber"

23 Publications

  • Page 1 of 1

Drag-induced directionality switching of kinesin-5 Cin8 revealed by cluster-motility analysis.

Sci Adv 2021 Feb 5;7(6). Epub 2021 Feb 5.

Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel.

Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of directionality switching of mitotic kinesin-5 motors challenged the long-standing paradigm that individual kinesin motors are characterized by an intrinsic directionality. The underlying mechanism, however, remains unexplained. Here, we studied clustering-induced directionality switching of the bidirectional kinesin-5 Cin8. Based on the characterization of single-molecule and cluster motility, we developed a model that predicts that directionality switching of Cin8 is caused by an asymmetric response of its active motion to opposing forces, referred to as drag. The model shows excellent quantitative agreement with experimental data obtained under high and low ionic strength conditions. Our analysis identifies a robust and general mechanism that explains why bidirectional motor proteins reverse direction in response to seemingly unrelated experimental factors including changes in motor density and molecular crowding, and in multimotor motility assays.
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http://dx.doi.org/10.1126/sciadv.abc1687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864582PMC
February 2021

The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding.

Elife 2020 01 20;9. Epub 2020 Jan 20.

Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States.

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.
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http://dx.doi.org/10.7554/eLife.51131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015671PMC
January 2020

Synthetic-Evolution Reveals Narrow Paths to Regulation of the Mitotic Kinesin-5 Cin8.

Int J Biol Sci 2019 2;15(6):1125-1138. Epub 2019 May 2.

Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva, 84105, Israel.

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions, but some substrates maintain precise phosphosite positions over billions of years. Here, we examined the phosphoregulation of the kinesin-5, Cin8, using synthetic Cdk1-sites. We first analyzed the three native Cdk1 sites within the catalytic motor domain. Any single site conferred regulation, but to different extents. Synthetic sites were then systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites, 8 disrupted function; 19 were neutral, similar to the phospho-deficient variant; and only two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one was immediately adjacent to a native Cdk1 site. Only one novel site position resulted in phospho-regulation. This site was sampled elsewhere in evolution, but the synthetic version was inefficient in . This study shows that a single phosphorylation site can modulate complex spindle dynamics, but likely requires further evolution to optimally regulate the precise reaction cycle of a mitotic motor.
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http://dx.doi.org/10.7150/ijbs.30543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567808PMC
April 2020

The plus-tip tracking and microtubule stabilizing activities of Javelin-like regulate microtubule organization and cell polarity.

FEBS J 2019 10 17;286(19):3811-3830. Epub 2019 Jun 17.

Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel.

Cell polarity is essential for building cell asymmetry in all eukaryotic cells. Drosophila oocyte and bristle development require the newly characterized Spn-F protein complex, which includes Spn-F, IKKε, and Javelin-like (Jvl), to establish polarity. Jvl is a novel microtubule (MT)-associated protein; however, the mechanism by which it regulates MT organization is still unknown. We found that overexpression of Jvl stabilizes MTs and that jvl is needed for stable MT arrangement at the bristle tip and organization of the dynamic MT throughout the bristle shaft. At low levels of expression in cultured cells, Jvl behaved as a microtubule plus-end tracking protein. We demonstrated that Jvl physically interacts with the highly conserved MT end-binding protein 1 (EB1) using yeast two-hybrid and GST pull-down assays. This interaction is, however, dispensable for Jvl function in oocyte and bristle development. In addition, using a MT-binding assay, we saw that Jvl-C terminus directly binds to MTs. We also revealed that oocyte developmental arrest caused by Jvl overexpression in the germline can be rescued by mutations in its partners, spn-F and ikkε, suggesting that complex formation with Spn-F and IKKε is required for Jvl function in vivo. In summary, our results show that the microtubule plus-end tracking and stabilizing activities of Jvl are central for controlling cell polarity of oocytes and bristles.
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http://dx.doi.org/10.1111/febs.14944DOI Listing
October 2019

Bidirectional motility of kinesin-5 motor proteins: structural determinants, cumulative functions and physiological roles.

Cell Mol Life Sci 2018 05 3;75(10):1757-1771. Epub 2018 Feb 3.

Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PO Box 653, 84105, Beer-Sheva, Israel.

Mitotic kinesin-5 bipolar motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding antiparallel microtubules (MTs) apart within the mitotic spindle. Two recent studies have indicated that single molecules of Cin8, the Saccharomyces cerevisiae kinesin-5 homolog, are minus end-directed when moving on single MTs, yet switch directionality under certain experimental conditions (Gerson-Gurwitz et al., EMBO J 30:4942-4954, 2011; Roostalu et al., Science 332:94-99, 2011). This finding was unexpected since the Cin8 catalytic motor domain is located at the N-terminus of the protein, and such kinesins have been previously thought to be exclusively plus end-directed. In addition, the essential intracellular functions of kinesin-5 motors in separating spindle poles during mitosis can only be accomplished by plus end-directed motility during antiparallel sliding of the spindle MTs. Thus, the mechanism and possible physiological role of the minus end-directed motility of kinesin-5 motors remain unclear. Experimental and theoretical studies from several laboratories in recent years have identified additional kinesin-5 motors that are bidirectional, revealed structural determinants that regulate directionality, examined the possible mechanisms involved and have proposed physiological roles for the minus end-directed motility of kinesin-5 motors. Here, we summarize our current understanding of the remarkable ability of certain kinesin-5 motors to switch directionality when moving along MTs.
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http://dx.doi.org/10.1007/s00018-018-2754-7DOI Listing
May 2018

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

Cell Mol Life Sci 2017 09 28;74(18):3395-3412. Epub 2017 Apr 28.

Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PO Box 653, 84105, Beer-Sheva, Israel.

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.
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http://dx.doi.org/10.1007/s00018-017-2523-zDOI Listing
September 2017

A potential physiological role for bi-directional motility and motor clustering of mitotic kinesin-5 Cin8 in yeast mitosis.

J Cell Sci 2017 02 9;130(4):725-734. Epub 2017 Jan 9.

Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

The bipolar kinesin-5 Cin8 switches from minus- to plus-end-directed motility under various conditions The mechanism and physiological significance of this switch remain unknown. Here, we show that under high ionic strength conditions, Cin8 moves towards and concentrates in clusters at the minus ends of stable and dynamic microtubules. Clustering of Cin8 induces a switch from fast minus- to slow plus-end-directed motility and forms sites that capture antiparallel microtubules (MTs) and induces their sliding apart through plus-end-directed motility. In early mitotic cells with monopolar spindles, Cin8 localizes near the spindle poles at microtubule minus ends. This localization is dependent on the minus-end-directed motility of Cin8. In cells with assembled bipolar spindles, Cin8 is distributed along the spindle microtubules. We propose that minus-end-directed motility is required for Cin8 clustering near the spindle poles before spindle assembly. Cin8 clusters promote the capture of microtubules emanating from the neighboring spindle poles and mediate their antiparallel sliding. This activity is essential to maximize microtubule crosslinking before bipolar spindle assembly and to induce the initial separation of the spindle poles.
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http://dx.doi.org/10.1242/jcs.195040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339886PMC
February 2017

Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domain.

Sci Rep 2016 05 24;6:25597. Epub 2016 May 24.

Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.

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.
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http://dx.doi.org/10.1038/srep25597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877575PMC
May 2016

Mitotic slippage and expression of survivin are linked to differential sensitivity of human cancer cell-lines to the Kinesin-5 inhibitor monastrol.

PLoS One 2015 2;10(6):e0129255. Epub 2015 Jun 2.

Department of Chemistry, Faculty of Natural Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

The mitotic Kinesin-5 motor proteins crosslink and slide apart antiparallel spindle microtubules, thus performing essential functions in mitotic spindle dynamics. Specific inhibition of their function by monastrol-like small molecules has been examined in clinical trials as anticancer treatment, with only partial success. Thus, strategies that improve the efficiency of monastrol-like anticancer drugs are required. In the current study, we examined the link between sensitivity to monastrol and occurrence of mitotic slippage in several human cell-lines. We found that the rank of sensitivity to monastrol, from most sensitive to least sensitive, is: AGS > HepG2 > Lovo > Du145 ≥ HT29. We show correlation between the sensitivity of a particular cell-line to monastrol and the tendency of the same cell-line to undergo mitotic slippage. We also found that in the monastrol resistant HT29 cells, prolonged monastrol treatments increase mRNA and protein levels of the chromosomal passenger protein survivin. In contrast, survivin levels are not increased by this treatment in the monastrol-sensitive AGS cells. We further show that over-expression of survivin in the monastrol-sensitive AGS cells reduces mitotic slippage and increases resistance to monastrol. Finally, we show that during short exposure to monastrol, Si RNA silencing of survivin expression reduces cell viability in both AGS and HT29 cells. Our data suggest that the efficiency of anti-cancer treatment with specific kinesin-5 inhibitors may be improved by modulation of expression levels of survivin.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129255PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452773PMC
April 2016

Deletion of the Tail Domain of the Kinesin-5 Cin8 Affects Its Directionality.

J Biol Chem 2015 Jul 19;290(27):16841-50. Epub 2015 May 19.

From the Drittes Physikalisches Institut, Georg-August-Universität, 37077 Göttingen, Germany,

The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility.
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http://dx.doi.org/10.1074/jbc.M114.620799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505430PMC
July 2015

Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo.

J Cell Sci 2013 Sep 18;126(Pt 18):4147-59. Epub 2013 Jul 18.

Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

In this study, we examined the anaphase functions of the S. cerevisiae kinesin-5 homolog Kip1. We show that Kip1 is attached to the mitotic spindle midzone during late anaphase. This attachment is essential to stabilize interpolar microtubule (iMTs) plus-ends. By detailed examination of iMT dynamics we show that at the end of anaphase, iMTs depolymerize in two stages: during the first stage, one pair of anti-parallel iMTs depolymerizes at a velocity of 7.7 µm/minute; during the second stage, ∼90 seconds later, the remaining pair of iMTs depolymerizes at a slower velocity of 5.4 µm/minute. We show that upon the second depolymerization stage, which coincides with spindle breakdown, Kip1 follows the plus-ends of depolymerizing iMTs and translocates toward the spindle poles. This movement is independent of mitotic microtubule motor proteins or the major plus-end binding or tracking proteins. In addition, we show that Kip1 processively tracks the plus-ends of growing and shrinking MTs, both inside and outside the nucleus. The plus-end tracking activity of Kip1 requires its catalytic motor function, because a rigor mutant of Kip1 does not exhibit this activity. Finally, we show that Kip1 is a bi-directional motor: in vitro, at high ionic strength conditions, single Kip1 molecules move processively in the minus-end direction of the MTs, whereas in a multi-motor gliding assay, Kip1 is plus-end directed. The bi-directionality and plus-end tracking activity of Kip1, properties revealed here for the first time, allow Kip1 to perform its multiple functions in mitotic spindle dynamics and to partition the 2-micron plasmid.
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http://dx.doi.org/10.1242/jcs.125153DOI Listing
September 2013

Regulation of bi-directional movement of single kinesin-5 Cin8 molecules.

Bioarchitecture 2012 Feb;2(2):70-74

Kinesin-5 mechanoenzymes drive mitotic spindle dynamics as slow, processive microtubule (MT)-plus-end directed motors. Surprisingly, the Saccharomyces cerevisiae kinesin-5 Cin8 was recently found to be bi-directional: it can move processively in both directions on MTs. Two hypotheses have been suggested for the mechanism of the directionality switch: (1) single molecules of Cin8 are intrinsically minus-end directed, but mechanical coupling between two or more motors triggers the switch; (2) a single motor can switch direction, and "cargo binding" i.e., binding between two MTs triggers the switch to plus-end motility. Single-molecule fluorescence data we published recently, and augment here, favor hypothesis (2). In low-ionic-strength conditions, single molecules of Cin8 move in both minus- and plus-end directions. Fluorescence photo bleaching data rule out aggregation of Cin8 while they move in the plus and in the minus direction. The evidence thus points toward cargo regulation of directionality, which is likely to be related to cargo regulation in other kinesins. The molecular mechanisms of this regulation, however, remain to be elucidated.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383724PMC
http://dx.doi.org/10.4161/bioa.20395DOI Listing
February 2012

Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry.

EMBO J 2011 Nov 18;30(24):4942-54. Epub 2011 Nov 18.

Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Kinesin-5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly (∼25 nm/s) towards the midzone, but occasionally also faster (∼55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus-end relative sliding. Between parallel MTs, plus-end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic strength-dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.
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http://dx.doi.org/10.1038/emboj.2011.403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243633PMC
November 2011

Fine-tuning of the Msn2/4-mediated yeast stress responses as revealed by systematic deletion of Msn2/4 partners.

Mol Biol Cell 2011 Sep 14;22(17):3127-38. Epub 2011 Jul 14.

Departments of Life Science, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel.

The Msn2 and Msn4 transcription factors play major roles in the yeast general stress response by mediating the transcription of hundreds of genes. Despite extensive information on Msn2/4-mediated gene expression profiles, much less is known regarding the network of proteins that regulate its activity. Here we describe a systematic approach designed to examine the roles of 35 Msn2/4 partners in regulating Msn2/4 transcriptional activity in the face of four different environmental conditions. Our analysis indicates that single deletions of 26 Msn2/4 partners significantly affect Msn2/4 transcription activity under four different conditions. The low functional redundancy of the Msn2 regulatory network indicates that Msn2/4 activity is finely tuned by many of Msn2/4 partners to provide an optimized stress response through differential activation, nuclear localization, degradation, and chromatin remodeling. Our specific analysis of Msn2 activity showed that a relatively large number of partners act to suppress Msn2 activity under nonstress conditions through independent mechanisms, including cytoplasmic retention, proteosome-mediated Msn2 degradation, and chromatin remodeling. Such negative regulation is crucial to minimize the cost of uncontrolled stress response gene expression and ensures a high growth rate in the absence of stress.
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http://dx.doi.org/10.1091/mbc.E10-12-1007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164460PMC
September 2011

Phospho-regulation of kinesin-5 during anaphase spindle elongation.

J Cell Sci 2011 Mar;124(Pt 6):873-8

Department of Clinical Biochemistry, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva, 84105, Israel.

The kinesin-5 Saccharomyces cerevisiae homologue Cin8 is shown here to be differentially phosphorylated during late anaphase at Cdk1-specific sites located in its motor domain. Wild-type Cin8 binds to the early-anaphase spindles and detaches from the spindles at late anaphase, whereas the phosphorylation-deficient Cin8-3A mutant protein remains attached to a larger region of the spindle and spindle poles for prolonged periods. This localization of Cin8-3A causes faster spindle elongation and longer anaphase spindles, which have aberrant morphology. By contrast, the phospho-mimic Cin8-3D mutant exhibits reduced binding to the spindles. In the absence of the kinesin-5 homologue Kip1, cells expressing Cin8-3D exhibit spindle assembly defects and are not viable at 37°C as a result of spindle collapse. We propose that dephosphorylation of Cin8 promotes its binding to the spindle microtubules before the onset of anaphase. In mid to late anaphase, phosphorylation of Cin8 causes its detachment from the spindles, which reduces the spindle elongation rate and aids in maintaining spindle morphology.
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http://dx.doi.org/10.1242/jcs.077396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048887PMC
March 2011

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress.

Cell Mol Life Sci 2010 Jun 4;67(12):2025-38. Epub 2010 Mar 4.

Department of Life Sciences, Ben Gurion University of the Negev, P.O. Box 653, 84105, Beersheba, Israel.

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.
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http://dx.doi.org/10.1007/s00018-010-0308-8DOI Listing
June 2010

A similar cell-specific pattern of HOXA methylation in normal and in cancer tissues.

Epigenetics 2010 Jan 23;5(1):41-6. Epub 2010 Jan 23.

Department of Oncology, Assaf Harofeh Medical Center affiliated with Tel-Aviv University, Zerifin, Israel.

HOX genes are developmental genes that determine anterior-posterior embryonic pattern and govern the process of differentiation. Inappropriate expression of HOX genes has been implicated in developmental abnormalities and hematopoietic malignancies. In addition, HOX genes silencing by DNA methylation has been reported in cancers and related to disease aggressiveness and outcome. On the other hand, accumulating evidence suggests that epigenetic changes at HOX genes are linked to normal development and differentiation. To better understand the relationship between HOXA methylation and cancer, we analyzed the methylation pattern of HOXA genes in human primary breast and colon carcinomas, normal tissues, and normal white blood cells. Genome-wide methylation arrays of breast cancers and white blood cells demonstrated similar methylation patterns. Quantitative methylation analysis of seven representative HOXA genes revealed various levels of methylation in both normal tissues and cancers. Analysis of epithelial-enriched normal breast tissue and stroma indicated that the stroma was the major origin of HOXA methylation. Furthermore, in selected dense breast cancers, minimal increase in methylation of several HOXA genes did not correlate with the predominance of malignant epithelial cells in these tumors. Our results suggest that methylation of the HOXA cluster may be a normal developmental and cell type specific process rather than a cancer specific mechanism.
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http://dx.doi.org/10.4161/epi.5.1.10724DOI Listing
January 2010

Midzone organization restricts interpolar microtubule plus-end dynamics during spindle elongation.

EMBO Rep 2009 Apr 6;10(4):387-93. Epub 2009 Mar 6.

Department of Clinical Biochemistry, Ben-Gurion University of the Negev, PO Box 653, Beer Sheva 84105, Israel.

To study the dynamics of interpolar microtubules (iMTs) in Saccharomyces cerevisiae cells, we photobleached a considerable portion of the middle region of anaphase spindles in cells expressing tubulin-green fluorescent protein (GFP) and followed fluorescence recovery at the iMT plus-ends. We found that during anaphase, iMTs show phases of fast growth and shrinkage that are restricted to the iMT plus-ends. Our data indicate that iMT plus-end dynamics are regulated during mitosis, as fluorescence recovery was faster in intermediate anaphase (30 s) compared with long (100 s) and pre-anaphase (80 s) spindles. We also observed that deletion of Cin8, a microtubule-crosslinking kinesin-5 motor protein, reduced the recovery rate in anaphase spindles, indicating that Cin8 contributes to the destabilization of iMT plus-ends. Finally, we show that in cells lacking the midzone organizing protein Ase1, iMTs are highly dynamic and are exchangeable throughout most of their length, indicating that midzone organization is essential for restricting iMT dynamics.
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http://dx.doi.org/10.1038/embor.2009.7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2672901PMC
April 2009

Slk19-dependent mid-anaphase pause in kinesin-5-mutated cells.

J Cell Sci 2008 Aug 15;121(Pt 15):2529-39. Epub 2008 Jul 15.

Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

We examined spindle elongation in anaphase in Saccharomyces cerevisiae cells mutated for the kinesin-5 motor proteins Cin8 and Kip1. Cells were deleted for KIP1 and/or expressed one of two motor-domain Cin8 mutants (Cin8-F467A or Cin8-R196K, which differ in their ability to bind microtubules in vitro, with Cin8-F467A having the weakest ability). We found that, in kinesin-5-mutated cells, predominantly in kip1 Delta cin8-F467A cells, anaphase spindle elongation was frequently interrupted after the fast phase, resulting in a mid-anaphase pause. Expression of kinesin-5 mutants also caused an asymmetric midzone location and enlarged midzone size, suggesting that proper organization of the midzone is required for continuous spindle elongation. We also examined the effects of components of the FEAR pathway, which is involved in the early-anaphase activation of Cdc14 regulatory phosphatase, on anaphase spindle elongation in kip1 Delta cin8-F467A cells. Deletion of SLK19, but not SPO12, eliminated the mid-anaphase pause, caused premature anaphase onset and defects in DNA division during anaphase, and reduced viability in these cells. Finally, overriding of the pre-anaphase checkpoint by overexpression of Cdc20 also eliminated the mid-anaphase pause and caused DNA deformation during anaphase in kip1 Delta cin8-F467A cells. We propose that transient activation of the pre-anaphase checkpoint in kinesin-5-mutated cells induces a Slk19-dependent mid-anaphase pause, which might be important for proper DNA segregation.
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http://dx.doi.org/10.1242/jcs.022996DOI Listing
August 2008

Extracellular zinc and zinc-citrate, acting through a putative zinc-sensing receptor, regulate growth and survival of prostate cancer cells.

Carcinogenesis 2008 Sep 28;29(9):1692-700. Epub 2008 Feb 28.

Department of Morphology, Zlotowski Center for Neuroscience, Faculty of Health Sciences, Ben-Gurion University, PO Box 653, Beer Sheva 84105, Israel.

Prostate Zn(2+) concentrations are among the highest in the body, and a marked decrease in the level of this ion is observed in prostate cancer. Extracellular Zn(2+) is known to regulate cell survival and proliferation in numerous tissues. In spite of this, a signaling role for extracellular Zn(2+) in prostate cancer has not been established. In the present study, we demonstrate that prostate metastatic cells are impermeable to Zn(2+), but extracellular Zn(2+) triggers a metabotropic Ca(2+) rise that is also apparent in the presence of citrate. Employing fluorescent imaging, we measured this activity in androgen-insensitive metastatic human cell lines, PC-3 and DU-145, and in mouse prostate tumor TRAMP-1 cells but not in androgen-sensitive LNCaP cells. The Ca(2+) response was inhibited by Galphaq and phospholipase C (PLC) inhibitors as well as by intracellular Ca(2+) store depletion, indicating that it is mediated by a Gq-coupled receptor that activates the inositol phosphate (IP(3)) pathway consistent with the previously identified zinc-sensing receptor (ZnR). Zn(2+)-dependent extracellular signal-regulated kinase and AKT activation, as well as enhanced Zn(2+)-dependent cell growth and survival, were observed in PC-3 cells that exhibit ZnR activity, but not in a ZnR activity-deficient PC-3 subline. Interestingly, application of Zn(2+)-citrate (Zn(2+)Cit), at physiological concentrations, was followed by a profound functional desensitization of extracellular Zn(2+)-dependent signaling and attenuation of Zn(2+)-dependent cell growth. Our results indicate that extracellular Zn(2+) and Zn(2+)Cit, by triggering or desensitizing ZnR activity, distinctly regulate prostate cancer cell growth. Thus, therapeutic strategies based either on Zn(2+) chelation or administration of Zn(2+)Cit may be effective in attenuating prostate tumor growth.
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http://dx.doi.org/10.1093/carcin/bgn027DOI Listing
September 2008

Homotetrameric form of Cin8p, a Saccharomyces cerevisiae kinesin-5 motor, is essential for its in vivo function.

J Biol Chem 2006 Sep 7;281(36):26004-13. Epub 2006 Jul 7.

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.

Kinesin-5 motor proteins are evolutionarily conserved and perform essential roles in mitotic spindle assembly and spindle elongation during anaphase. Previous studies demonstrated a specialized homotetrameric structure with two pairs of catalytic domains, one at each end of a dumbbell-shaped molecule. This suggests that they perform their spindle roles by cross-linking and sliding antiparallel spindle microtubules. However, the exact kinesin-5 sequence elements that are important for formation of the tetrameric complexes have not yet been identified. In addition, it has not been demonstrated that the homotetrameric form of these proteins is essential for their biological functions. Thus, we investigated a series of Saccharomyces cerevisiae Cin8p truncations and internal deletions, in order to identify structural elements in the Cin8p sequence that are required for Cin8p functionality, spindle localization, and multimerization. We found that all variants of Cin8p that are functional in vivo form tetrameric complexes. The first coiled-coil domain in the stalk of Cin8p, a feature that is shared by all kinesin-5 homologues, is required for its dimerization, and sequences in the last part of the stalk, specifically those likely involved in coiled-coil formation, are required for Cin8p tetramerization. We also found that dimeric forms of Cin8p that are nonfunctional in vivo can nonetheless bind to microtubules. These findings suggest that binding of microtubules is not sufficient for the functionality of Cin8p and that microtubule cross-linking by the tetrameric complex is essential for Cin8p mitotic functions.
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http://dx.doi.org/10.1074/jbc.M604817200DOI Listing
September 2006

Carotenoids activate the antioxidant response element transcription system.

Mol Cancer Ther 2005 Jan;4(1):177-86

Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Epidemiologic studies have found an inverse association between consumption of tomato products and the risk of certain types of cancers. However, the mechanisms underlying this relationship are not completely understood. One mechanism that has been suggested is induction of phase II detoxification enzymes. Expression of phase II enzymes is regulated by the antioxidant response element (ARE) and the transcription factor Nrf2 (nuclear factor E2-related factor 2). In this study, we determined the role of this transcription system in the induction of phase II enzymes by carotenoids. We found that in transiently transfected cancer cells, lycopene transactivated the expression of reporter genes fused with ARE sequences. Other carotenoids such as phytoene, phytofluene, beta-carotene, and astaxanthin had a much smaller effect. An increase in protein as well as mRNA levels of the phase II enzymes NAD(P)H:quinone oxidoreductase and gamma-glutamylcysteine synthetase was observed in nontransfected cells after carotenoid treatment. Ethanolic extract of lycopene containing unidentified hydrophilic derivatives of the carotenoid activated ARE with similar potency to lycopene. The potency of the carotenoids in ARE activation did not correlate with their effect on intracellular reactive oxygen species and reduced glutathione level, which may indicate that ARE activation is not solely related to their antioxidant activity. Nrf2, which is found predominantly in the cytoplasm of control cells, translocated to the nucleus after carotenoid treatment. Interestingly, part of the translocated Nrf2 colocalized with the promyelocytic leukemia protein in the promyelocytic leukemia nuclear bodies. The increase in phase II enzymes was abolished by a dominant-negative Nrf2, suggesting that carotenoid induction of these proteins depends on a functional Nrf2 and the ARE transcription system.
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January 2005

"Bread madness" revisited: screening for specific celiac antibodies among schizophrenia patients.

Eur Psychiatry 2004 Aug;19(5):311-4

Department of Family Medicine, Sial Research Center for Family Medicine and Primary Care, Division of Community Health, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.

Purpose: A possible association between gluten consumption and schizophrenia has been reported. The objective was to compare patients with chronic schizophrenia and matched controls for sociodemographic variables, prevalence of celiac-specific anti-endomysial antibodies and disease-related variables.

Subjects And Methods: The study group was comprised of 50 consecutive patients diagnosed with schizophrenia, 18 years of age and older attending the out-patient clinic of the Mental Health Center in Beer-Sheva, Israel. The control group was comprised of mentally normal volunteers who came to primary care clinics for blood tests unrelated to gastrointestinal tract complaints and who were not diagnosed with celiac disease. Known celiac patients and those who refused to participate, did non-speak Hebrew or were incoherent were excluded from the study. All participants in both groups underwent a blood test for anti-endomysial IgA antibody and completed a questionnaire.

Results: Each group was comprised of 50 participants. There were no significant differences between the groups in gender, BMI or country of birth. The mean age of the study group was significantly higher than the controls. All tests for anti-endomysial antibody in both groups were negative.

Discussion And Conclusions: In contrast to previous reports, we found no evidence for celiac disease in patients with chronic schizophrenia as manifested by the presence of serum IgA anti-endomysial antibodies. It is unlikely that there is an association between gluten sensitivity and schizophrenia.
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http://dx.doi.org/10.1016/j.eurpsy.2004.06.003DOI Listing
August 2004