The Role of Hklp2 in the Stabilization and Maintenance of Spindle Bipolarity

SummarySpindle bipolarity relies on a fine balance of forces exerted by various molecular motors [1–4]. In most animal cells, spindle bipolarity requires sustained outward forces to push the spindle poles apart, an activity that is provided by Eg5, a conserved homotetrameric plus-end-directed kinesin that crosslinks and slides antiparallel microtubules apart [5]. These pushing forces are balanced by inward minus-end-directed forces. Impairing both Eg5 and dynein restores the formation of functional bipolar spindles [4], although the mechanism at play is far from clear. The current model also fails to explain why in some systems Eg5 inhibition does not promote bipolar spindle collapse [6, 7] or why increasing Eg5 levels does not interfere with bipolar spindle assembly [8]. Moreover, the C. elegans Eg5 ortholog is not required for bipolar spindle formation [9]. We show here that the kinesin Hklp2 participates in the assembly and stabilization of the bipolar spindle. Hklp2 localizes to the mitotic microtubules in a TPX2-dependent manner and to the chromosomes through Ki67. Our data indicate that its mechanism of action is clearly distinct from and complementary to that of Eg5, providing an additional understanding of the mechanism driving the formation and maintenance of the bipolar spindle

Mutations in fission yeast Cut15, an importin α homolog, lead to mitotic progression without chromosome condensation

AbstractChromosome condensation is a major mitotic event [1]. Fission yeast mutations in topoisomerase II and condensin subunits produce the characteristic ‘cut’ phenotypes, in which the septum bisects the nuclear material in the absence of normal condensation and sister chromatid separation [2]. We show here that the same condensation defect is produced in cut15 temperature-sensitive mutants at the restrictive temperature (36 ° C). The gene product of cut15+ is, surprisingly, very similar to importin α[3,4], which binds proteins containing a nuclear localization signal (NLS) and forms the heterodimer with importin β that mediates translocation through the nuclear pore complex [5]. We show that in a nuclear import assay, purified Cut15 protein behaved identically to mammalian importin α but mutant Cut15 did not. Mutant Cut15 failed to bind an NLS-containing protein in vitro but could still bind importin β. Unexpectedly, however, NLS proteins were imported into the nucleus in cut15 mutants. Cut15 is thus essential for mitotic chromosome condensation, but its role in nuclear import might be dispensable. Green fluorescent protein (GFP)-tagged Cut15 was enriched within the nucleus specifically during prometaphase–metaphase, so the interaction of Cut15 with nuclear NLS proteins during mitosis might be important for condensation

The 70-kD heat shock cognate protein (hsc70) facilitates the nuclear export of the import receptors

Transport receptors of the importin β family continuously shuttle between the nucleus and cytoplasm. We previously reported that the nuclear export of importin β involves energy-requiring step(s) in living cells. Here, we show that the in vitro nuclear export of importin β also requires energy input. Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin β. Further purification revealed that the active component in the absorbed fraction was a 70-kD heat shock cognate protein (hsc70). The addition of recombinant hsc70, but not an ATPase-deficient hsc70 mutant, to the depleted cytosol restored the export activity. In living cells, depletion of hsc70 caused the significant nuclear accumulation of importin β. These effects of hsc70 were observed in the nuclear export of importin β, but also for other import receptors, transportin and importin α. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins

Importin-β and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid

Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-α/β transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-α/β was efficiently targeted to mitotic chromosomes. The addition of Ran–guanosine diphosphate and an energy source, which generates Ran–guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-β–mediated chromosome loading of hKid. Our results indicate that the association of importin-β and -α with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP–mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor–mediated mechanism for targeting proteins to mitotic chromosomes

Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain

Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left–right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1–4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin α1/α6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS