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author:("heber, Larisa")
1.  Regulation of bi-directional movement of single kinesin-5 Cin8 molecules 
Bioarchitecture  2012;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.
PMCID: PMC3383724  PMID: 22754632
Saccharomyces cerevisiae Cin8; kinesin directionality; kinesin-5; microtubules; mitosis
2.  Fine-tuning of the Msn2/4–mediated yeast stress responses as revealed by systematic deletion of Msn2/4 partners 
Molecular Biology of the Cell  2011;22(17):3127-3138.
Msn2 and Msn4 transcription factors play a major role in yeast response to a variety of stress conditions. A systematic approach to the identification of Msn2/4 activators or suppressors shows that the majority of the Msn2 protein regulatory network acts to fine-tune its activity following yeast exposure to diverse stress conditions.
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.
PMCID: PMC3164460  PMID: 21757539
3.  Phospho-regulation of kinesin-5 during anaphase spindle elongation 
Journal of Cell Science  2011;124(6):873-878.
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.
PMCID: PMC3048887  PMID: 21378308
Cdk1; Cin8; Kinesin-5; Microtubules; Mitosis
4.  Novel Roles for Saccharomyces cerevisiae Mitotic Spindle Motors 
The Journal of Cell Biology  1999;147(2):335-350.
The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.
PMCID: PMC2174232  PMID: 10525539
kinesin; dynein; motor proteins; microtubules; mitotic spindle

Results 1-4 (4)