Publications by authors named "Alina Goldstein"

4 Publications

  • Page 1 of 1

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

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

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