Phospho-regulation of kinesin-5 during anaphase spindle elongation

Abstract

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.

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

“…In contrast to Cin8, which detaches from the spindle midzone at late anaphase (Avunie-Masala et al, 2011), Kip1 remained attached to this region, during late anaphase stages. This difference in localization was confirmed by co-expressing Kip1-3GFP and Cin8-tdTomato: in the majority of lateanaphase spindles (.6 mm), Kip1 was enriched at the midzone compared to Cin8 (Fig.…”

“…The differences in regulation of Cin8 and Kip1 at this stage may be due to the presence of the long loop 8 of Cin8, possibly exerting its timely function through phosphorylation of two Cdk1 phosphorylation sites, which are absent in Kip1. Detachment of Cin8 from the spindle slows down spindle elongation rate (Avunie-Masala et al, 2011) giving rise to the second, slower stage of anaphase spindle elongation, mediated by the motor activity of Kip1 (Straight et al, 1998). Kip1 remains attached to the midzone region until the spindle breaks down.…”

“…Live-cell imaging was done using a spinning-disc confocal microscope at the Ilse Katz Institute for Nanoscale Science and Technology (Zeiss Axiovert 200M, UltraView ESR, Perkin Elmer, UK), equipped with a FRAP module, as previously described (Avunie-Masala et al, 2011;Fridman et al, 2009;Gerson-Gurwitz et al, 2011). Z-stacks of 0.2-0.3 mm separations were acquired with frame time of 2-4 seconds/frame for in vivo imaging of MT dynamics, and 60 seconds/frame for spindle elongation measurements.…”

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

“…One of the major factors that govern the mitotic spindle dynamics is the function of kinesin-5 motor proteins (1)(2)(3)(4)(5), which share homotetrameric structure, with two catalytic domains located at the opposite ends of a coiled-coil stalk (6,7). This bipolar structure enables kinesin-5 motors to cross-link and slide apart antiparallel microtubules (MTs) 4 (8,9), emanating from the opposing spindle poles, in yeast spindle pole bodies (SPBs) (10 -12).…”

“…This bipolar structure enables kinesin-5 motors to cross-link and slide apart antiparallel microtubules (MTs) 4 (8,9), emanating from the opposing spindle poles, in yeast spindle pole bodies (SPBs) (10 -12). Kinesin-5 motors are believed to thus provide the necessary SPBseparating force to perform essential functions in spindle assembly and maintenance of the bipolar spindle structure (10,11) and anaphase B spindle elongation (1)(2)(3)(4)13).…”