Distinct Kinesin-14 mitotic mechanisms in spindle bipolarity

Abstract

Kinesin-like proteins are integral to formation and function of a conserved mitotic spindle apparatus that directs chromosome segregation and precedes cell division. Ubiquitous to the mechanism of spindle assembly and stability are balanced Kinesin-5 promoting and Kinesin-14 opposing forces. Distinct Kinesin-14 roles in bipolarity in eukaryotes have not been shown, but are suggested by gamma-tubulin-based pole interactions that affect establishment and by microtubule cross-linking and sliding that maintain bipolarity and spindle length. Distinct roles also imply specialized functional domains. By cross-species analysis of compatible mechanisms in establishing mitotic bipolarity we demonstrate that Kinesin-14 human HSET (HsHSET) functionally replaces Schizosaccharomyces pombe Pkl1 and its action is similarly blocked by mutation in a Kinesin-14 binding site on gamma-tubulin. Drosophila DmNcd localizes preferentially to bundled interpolar microtubules in fission yeast and does not replace SpPkl1. Analysis of twenty-six Kinesin-14 derivatives, including Tail, Stalk or Neck-Motor chimeras, for spindle localization, spindle assembly and mitotic progression defined critical domains. The Tail of SpPkl1 contains functional elements enabling its role in spindle assembly that are distinct from but transferable to DmNcd, whereas HsHSET function utilizes both Tail and Stalk features. Our analysis is the first to demonstrate distinct mechanisms between SpPkl1 and DmNcd, and reveal that HsHSET shares functional overlap in spindle pole mechanisms.

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation

“…Mouse KIFC1 has tail domain sequences that target it to membrane-bound organelles (Zhang and Sperry, 2004); the tail domain of Pkl1 direct it to γ-tubulin (Olmsted et al, 2013(Olmsted et al, , 2014. Additional studies in which chimeric kinesin-14 proteins of human HSET, Drosophila Ncd and fission yeast Pkl1 were generated also support this paradigm and confirm that tail domain sequences orchestrate function (Simeonov et al, 2009). …”

“…The function of spindle pole organization and microtubule nucleation does not need to be distinct, and some kinesin-14 members, such as HSET, might possess both capabilities of spindle pole organization and microtubule nucleation (Walczak et al, 1997;Simeonov et al, 2009). It is worth noting that in meiotic cells and in somatic cells, spindles can nucleate around chromosomes in the presence of a Ran gradient and this may explain in part the evolutionary need for kinesin-14 members to have dual roles with γ-tubulin complexes at the different Ran-GTP concentrations (Khodjakov et al, 2000;Khodjakov and Rieder, 2001).…”

“…In fibroblast cells or other cell types with a radial array of microtubules, almost all microtubules cluster with their minus-ends embedded in the centrosome (Desai and Mitchison, 1997). In several differentiated cells, including epithelial cells or neurons, non-centrosomal microtubules predominate and microtubules are arranged in parallel or anti-parallel to one another (Akhmanova and Hoogenraad, 2015;Keating and Borisy, 1999;Simeonov et al, 2009;Yvon and Wadsworth, 1997).…”

“…In contrast to plus-end-directed kinesins, kinesin-14 motors are specific minus-end-directed motors; they utilize the chemical energy of ATP hydrolysis to move along microtubules from their plus-to their minus-end (Friel and Howard, 2012;Schnitzer and Block, 1997;Shimizu et al, 1995). The kinesin-14 family is ubiquitous to all eukaryotes, with typically two to three members present that are different in their structure and function (Olmsted et al, 2015;Simeonov et al, 2009).…”

Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation

“…Similarly, we observed that anaphase cells in logarithmically growing cultures lacking CaKar3 and CaCik1 have spindles that are indistinguishable from those of the wild type in static images and that these do not collapse during anaphase. The S. pombe kinesin-14 counterpart to ScKar3/Vik1, named Pkl1, also displays SPB localization (91), and a recent study suggested that S. pombe Pkl1 and the kinesin-5 homolog Cut7 antagonistically regulate microtubule nucleation from the ␥-TuRC complex (92). This is intriguing because GFP-labeled C. albicans Kip1 (kinesin-5) also localizes prominently to the SPB, with a smaller quantity localizing with the spindle itself (10).…”