Vladimir Fridman

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

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.

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

Summary 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.

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

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.

Midzone organization restricts interpolar microtubule plus‐end dynamics during spindle elongation

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.

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

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.

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

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.

Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein

Abstract.S. cerevisiae anaphase spindle elongation is accomplished by the overlapping function of dynein and the kinesin-5 motor proteins, Cin8 and Kip1. Cin8 and dynein are synthetically lethal, yet the arrest phenotypes of cells eliminated for their function had not been identified. We found that at a non-permissive temperature, dyn1Δ cells that carry a temperature-sensitive cin8 – 3 mutation arrest at mid-anaphase with a unique phenotype, which we named TAN (two microtubule asters in one nucleus). These cells enter anaphase, but fail to proceed through the slow phase of anaphase B. At a permissive temperature, dyn1Δ, cin8 – 3 or dyn1Δcin8 – 3 cells exhibit perturbed spindle midzone morphologies, with dyn1Δcin8 – 3 anaphase spindles also being profoundly bent and nonrigid. Sorbitol, which has been suggested to stabilize microtubules, corrects these defects and suppresses the TAN phenotype. We conclude that dynein and Cin8 cooperate in anaphase midzone organization and influence microtubule dynamics, thus enabling progression through the slow phase of anaphase B.

Deletion of the tail domain of the kinesin-5 Cin8 affects its directionality

Background: Single molecules of the kinesin-5 Cin8 were previously demonstrated to be minus-end-directed under high-ionic-strength conditions. Results: Under high-ionic-strength conditions, Cin8 lacking the tail domain is bidirectional. Conclusion: The tail domain is one of the factors that regulate Cin8 directionality. Significance: An important structural element was identified that regulates the directionality of kinesin-5 motors. 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.