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

Abstract

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 kip1Δ 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 kip1Δ 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 kip1Δ 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.

Slk19p of Saccharomyces cerevisiae Regulates Anaphase Spindle Dynamics Through Two Independent Mechanisms

“…Slk19p, but not Spo12p, was previously demonstrated to be required for a midanaphase pause that could be detected in kinesin-5 mutants (Movshovich et al 2008). This is probably the same anaphase pause that is apparent in the analysis of our wild-type strains and wild-type strains used by others (Kahana et al 1995;Yeh et al 1995).…”

“…Strains lacking SLK19 exhibit multiple spindle defects including short mitotic spindles, increased numbers of astral microtubules, and fragile anaphase spindles (Zeng et al 1999;Sullivan et al 2001;Gardner et al 2008). These and other data suggest that Slk19p is a plus-end microtubule stabilizer (Zeng et al 1999;Movshovich et al 2008).…”

“…First, Slk19p mediates a pause during anaphase spindle elongation in kinesin-5 motor mutants (cin8-FA kip1D) (Movshovich et al 2008). Second, deletions of SLK19 result in more fragile or less forceful spindles; in slk19D kar3-ts double mutants (but less so in kar3-ts single mutants) the spindles collapse after a shift to the nonpermissive temperature (Zeng et al 1999), and slk19 mutants, like bim1, kar3, and ase1 mutants, are less able than wild-type cells to break a dicentric chromosome (Gardner et al 2008).…”

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

“…The function of kinesin-5 motors in spindle assembly (Blangy et al, 1995;Mayer et al, 1999;Saunders and Hoyt, 1992) and in anaphase spindle elongation (Movshovich et al, 2008;Saunders et al, 1995;Straight et al, 1998) is attributed to their ability to provide plus-end-directed motility and outwardly directed pushing forces between overlapping antiparallel MTs of the spindle . Thus, the role of the minus-enddirected activity of the S. cerevisiae kinesin-5 motors is less obvious.…”

“…At early-mid anaphase, during the first fast stage of anaphase spindle elongation, both Cin8 and Kip1 localize to the spindle with Cin8 contributing the majority of the SPB separation force (Movshovich et al, 2008;Saunders et al, 1995;Straight et al, 1998). At mid-late anaphase, Cin8 detaches from the spindles due to phosphorylation of Cdk1 targets located in its motor domain (Avunie-Masala et al, 2011) while Kip1 remains attached to the spindle midzone (Fig.…”