Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7

Abstract

ABSTRACT. Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubulebinding properties of this protein in vitro.

Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling

“…Three classes of motors and crosslinkers assemble the spindle ( Figure 1A,B). Kinesin-5 motors (representing Cut7) move bidirectionally on MTs [86][87][88][89], with plus-end directed movement on antiparallel MTs exerting force to slide apart the SPBs. Kinesin-14 motors (representing Pkl1 and Klp2) crosslink MTs and one head walks toward the MT minus-ends, aligning MTs and exerting force that shortens the spindle [13][14][15][90][91][92][93].…”

CyLaKS: the Cytoskeleton Lattice-based Kinetic Simulator

“…Problems including short-range interactions between motors [37,38] and motor response to patchy obstacles [39] are straighforward to simulate. Closely related are changes in motor behavior due to crowding, both crowding along the filament lattice [40][41][42][43][44][45]54] or due to crowders in solution [55], and motor direction switching [50][51][52][53][54]. Changes in the filament lattice, for example due to heterogeneous isoforms or post-translational modification [110][111][112] or lattice structure changes or defects [59][60][61][62][63][64] can be modeled in CyLaKS.…”

“…Kinesins that regulate microtubule length and dynamics typically show altered motility at microtubule ends [40][41][42][43][44][45][46][47][48][49]. Some kinesin-5 motors can switch their direction of motion along microtubules [50][51][52][53][54], an effect that is not well understood but may be regulated by crowding on the microtubule lattice [54]. Kinesin-1 stepping can be slowed in the presence of crowding molecules in solution, that appear to hinder diffusion of the motor domain [55].…”

Mechanisms by Which Kinesin-5 Motors Perform Their Multiple Intracellular Functions

“…Following the first reports on Cin8 bidirectionality, the kinesin-5 homologs S. cerevisiae Kip1, and S. pombe Cut7 were also reported to be bidirectional and exhibit switchable directionality under certain in vitro experimental conditions [35,36]. Subsequent work revealed intramolecular domains that affect the directionality of these three kinesin-5 motors [10], such as the N-terminal non-motor extension [141,142], the C-terminal tail domain [55], and the large insert in the loop 8 within the catalytic domain [33], which was also demonstrated to induce non-canonical binding of Cin8 to MTs [118]. Phosphorylation of three cyclin-dependent kinase 1 (Cdk1) sites in the catalytic domain of Cin8, which regulates its intracellular activity [58,60,61,143], with two of these sites being located in large loop 8, was also shown to affect Cin8 directionality [144].…”