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Bipolar spindle organization is essential for the faithful segregation of chromosomes during cell division. This organization relies on the collective activities of motor proteins. The minus-end-directed dynein motor complex generates spindle inward forces and plays a major role in spindle pole focusing. The dynactin complex regulates many dynein functions, increasing its processivity and force production. Here, we show that DnaJB6 is a novel RanGTP-regulated protein. It interacts with the dynactin subunit p150Glued (also known as DCTN1) in a RanGTP-dependent manner specifically in M-phase, and promotes spindle pole focusing and dynein force generation. Our data suggest a novel mechanism by which RanGTP regulates dynein activity during M-phase., Summary: DnaJB6 is a novel RanGTP-regulated protein that appears to play an important role in dynein-dependent spindle organization and spindle assembly.

During eukaryote cell division, molecular motors are crucial regulators of microtubule organization, spindle assembly, chromosome segregation and intracellular transport. The kinesin-14 motors are evolutionarily conserved minus-end-directed kinesin motors that occur in diverse organisms from simple yeasts to higher eukaryotes. Members of the kinesin-14 motor family can bind to, crosslink or slide microtubules and, thus, regulate microtubule organization and spindle assembly. In this Commentary, we present the common subthemes that have emerged from studies of the molecular kinetics and mechanics of kinesin-14 motors, particularly with regard to their non-processive movement, their ability to crosslink microtubules and interact with the minus- and plus-ends of microtubules, and with microtubule-organizing center proteins. In particular, counteracting forces between minus-end-directed kinesin-14 and plus-end-directed kinesin-5 motors have recently been implicated in the regulation of microtubule nucleation. We also discuss recent progress in our current understanding of the multiple and fundamental functions that kinesin-14 motors family members have in important aspects of cell division, including the spindle pole, spindle organization and chromosome segregation.

RanGTP is important for chromosome-dependent spindle assembly in Xenopus extracts. Here we report on experiments to determine the role of the Ran pathway on microtubule dynamics in Drosophila oocytes and embryos. Females expressing a dominant-negative form of Ran have fertility defects, suggesting that RanGTP is required for normal fertility. This is not, however, because of a defect in acentrosomal meiotic spindle assembly. Therefore, RanGTP does not appear to be essential or sufficient for the formation of the acentrosomal spindle. Instead, the most important function of the Ran pathway in spindle assembly appears to be in the tapering of microtubules at the spindle poles, which might be through regulation of proteins such as TACC and the HURP homolog, Mars. One consequence of this spindle organization defect is an increase in the nondisjunction of achiasmate chromosomes. However, the meiotic defects are not severe enough to cause the decreased fertility. Reductions in fertility occur because RanGTP has a role in microtubule assembly that is not directly nucleated by the chromosomes. This includes microtubules nucleated from the sperm aster, which are required for pronuclear fusion. We propose that following nuclear envelope breakdown, RanGTP is released from the nucleus and creates a cytoplasm that is activated for assembling microtubules, which is important for processes such as pronuclear fusion. Around the chromosomes, however, RanGTP might be redundant with other factors such as the chromosome passenger complex.

Ma N., Matsunaga S., Morimoto A., et al. The nuclear scaffold protein SAF-A is required for kinetochore-microtubule attachment and contributes to the targeting of Aurora-A to mitotic spindles. Journal of Cell Science, 124, 3, 394. https://doi.org/10.1242/jcs.063347., Segregation of chromosomes during cell division requires correct formation of mitotic spindles. Here, we show that a scaffold attachment factor A (SAF-A), also known as heterogeneous nuclear ribonucleoprotein-U, contributes to the attachment of spindle microtubules (MTs) to kinetochores and spindle organization. During mitosis, SAF-A was localized at the spindles, spindle midzone and cytoplasmic bridge. Depletion of SAF-A by RNA interference induced mitotic delay and defects in chromosome alignment and spindle assembly. We found that SAF-A specifically co-immunoprecipitated with the chromosome peripheral protein nucleolin and the spindle regulators Aurora-A and TPX2, indicating that SAF-A is associated with nucleolin and the Aurora-A-TPX2 complex. SAF-A was colocalized with TPX2 and Aurora-A in spindle poles and MTs. Elimination of TPX2 or Aurora-A from cells abolished the association of SAF-A with the mitotic spindle. Interestingly, SAF-A can bind to MTs and contributes to the targeting of Aurora-A to mitotic spindle MTs. Our finding indicates that SAF-A is a novel spindle regulator that plays an essential role in kinetochore-MT attachment and mitotic spindle organization.

End-binding 1 (EB1) proteins are evolutionarily conserved plus-end-tracking proteins that localize to growing microtubule plus ends where they regulate microtubule dynamics and interactions with intracellular targets. Animal EB1 proteins have acidic C-terminal tails that might induce an autoinhibitory conformation. Although EB1 proteins with the same structural features occur in plants (EB1a and EB1b in Arabidopsis thaliana), a variant form (EB1c) is present that lacks the characteristic tail. We show that in Arabidopsis the tail region of EB1b, but not of EB1c, inhibits microtubule assembly in vitro. EB1a and EB1b form heterodimers with each other, but not with EB1c. Furthermore, the EB1 genes are expressed in various cell types of Arabidopsis, but the expression of EB1c is particularly strong in the meristematic cells where it is targeted to the nucleus by a nuclear localization signal in the C-terminal tail. Reduced expression of EB1c compromised the alignment of spindle and phragmoplast microtubules and caused frequent lagging of separating chromosomes at anaphase. Roots of the eb1c mutant were hypersensitive to a microtubule-disrupting drug and complete rescue of the mutant phenotype required the tail region of EB1c. These results suggest that a plant-specific EB1 subtype has evolved to function preferentially on the spindle microtubules by accumulating in the prophase nucleus.

The formation of the mitotic spindle is controlled by the microtubule organizing activity of the centrosomes and by the effects of chromatin-associated Ran-GTP on the activities of spindle assembly factors. In this study we show that Mars, a Drosophila protein with sequence similarity to vertebrate hepatoma upregulated protein (HURP), is required for the attachment of the centrosome to the mitotic spindle. More than 80% of embryos derived from mars mutant females do not develop properly due to severe mitotic defects during the rapid nuclear divisions in early embryogenesis. Centrosomes frequently detach from spindles and from the nuclear envelope and nucleate astral microtubules in ectopic positions. Consistent with its function in spindle organization, Mars localizes to nuclei in interphase and associates with the mitotic spindle, in particular with the spindle poles, during mitosis. We propose that Mars is an important linker between the spindle and the centrosomes that is required for proper spindle organization during the rapid mitotic cycles in early embryogenesis.

In most species, oocytes lack centrosomes. Accurate meiotic spindle assembly and chromosome segregation – essential to prevent miscarriage or developmental defects – thus occur through atypical mechanisms that are not well characterized. Using quantitative in vitro and in vivo functional assays in the C. elegans oocyte, we provide novel evidence that the kinesin-13 KLP-7 promotes destabilization of the whole cellular microtubule network. By counteracting ectopic microtubule assembly and disorganization of the microtubule network, this function is strictly required for spindle organization, chromosome segregation and cytokinesis in meiotic cells. Strikingly, when centrosome activity was experimentally reduced, the absence of KLP-7 or the mammalian kinesin-13 protein MCAK (KIF2C) also resulted in ectopic microtubule asters during mitosis in C. elegans zygotes or HeLa cells, respectively. Our results highlight the general function of kinesin-13 microtubule depolymerases in preventing ectopic, spontaneous microtubule assembly when centrosome activity is defective or absent, which would otherwise lead to spindle microtubule disorganization and aneuploidy.

Centrosomes represent the major microtubule organizing centres (MTOCs) of animal somatic cells and orchestrate bipolar spindle assembly during mitotic cell division. In meiotic cells, the kinesin HSET compensates for the lack of centrosomes by focusing acentrosomal MTOCs into two spindle poles. By clustering multiple centrosomes into two spindle poles, HSET also mediates bipolar mitosis in cancer cells with supernumerary centrosomes. However, although dispensable in non-transformed human cells, the role of HSET in cancer cells with two centrosomes has remained elusive. In this study, we demonstrate that HSET is required for proper spindle assembly, stable pole-focusing and survival of cancer cells irrespective of normal or supernumerary centrosome number. Strikingly, we detected pronounced acentrosomal MTOC structures in untreated mitotic cancer cells. While in most cancer cells these acentrosomal MTOCs were rapidly incorporated into the assembling bipolar spindle, some cells eventually established bipolar spindles with acentrosomal poles and free centrosomes. These observations demonstrate that acentrosomal MTOCs were functional and that both centrosomal and acentrosomal mechanisms were required for bipolar spindle organization. Our study shows that HSET is critical for clustering acentrosomal and centrosomal MTOCs during spindle formation in human cancer cells with two bona fide centrosomes. Furthermore, we show that in checkpoint-defective cancer cells, acentrosomal spindle formation and HSET-dependence are partially mediated by a constitutive activation of the DNA damage response. In summary, we propose that acentrosomal spindle assembly mechanisms are hyperactive in cancer cells and promote HSET, a key driver of acentrosomal spindle organization, as an attractive target for cancer therapy.

The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase–anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.

The organization of the microtubule cytoskeleton depends crucially on crosslinking motors that arrange microtubules in space. Kinesin-5 is such an essential motile crosslinker. It is unknown whether its organizing capacity during bipolar spindle formation depends on its characteristic kinetic properties, or whether simply crosslinking combined with any plus-end-directed motility is sufficient for its function in a physiological context. To address this question, we replaced the motor domain of Xenopus Kinesin-5 by motor domains of kinesins belonging to other kinesin subfamilies, without changing the overall architecture of the molecule. This generated novel microtubule crosslinkers with altered kinetic properties. The chimeric crosslinkers mislocalized in spindles and consequently caused spindle collapse into tightly bundled microtubule arrays. This demonstrates that plus-end directionality and microtubule crosslinking are not the only characteristics required for proper functioning of Kinesin-5 during spindle assembly in Xenopus egg extract. Instead, its motor domain properties appear to be fine-tuned for the specific function of this kinesin.

Ma N., Matsunaga S., Takata H., et al. Nucleolin functions in nucleolus formation and chromosome congression. Journal of Cell Science, 120, 12, 2091. https://doi.org/10.1242/jcs.008771., A complex structure, designated the chromosome periphery, surrounds each chromosome during mitosis. Although several proteins have been shown to localize to the chromosome periphery, their functions during mitosis remain unclear. Here, we used a combination of high-resolution microscopy and RNA-interference-mediated depletion to study the functions of nucleolin, a nucleolar protein localized at the chromosome periphery, in interphase and mitosis. During mitosis, nucleolin was localized in the peripheral region including the vicinity of the outer kinetochore of chromosomes. Staining with an antibody specific for nucleolin phosphorylated by CDC2 revealed that nucleolin was also associated with the spindle poles from prometaphase to anaphase. Nucleolin depletion resulted in disorganization of the nucleoli at interphase. Furthermore, nucleolin-depleted cells showed a prolonged cell cycle with misaligned chromosomes and defects in spindle organization. The misaligned chromosomes showed syntelic kinetochore-microtubule attachments with reduced centromere stretching. Taken together, our results indicate that nucleolin is required for nucleolus formation, and is also involved in chromosome congression and spindle formation.

Astrin is a mitotic-spindle-associated protein expressed in most human cell lines and tissues. However, its functions in spindle organization and mitosis have not yet been determined. Sequence analysis revealed that astrin has an N-terminal globular domain and an extended coiled-coil domain. Recombinant astrin was purified and characterized by CD spectroscopy and electron microscopy. Astrin showed parallel dimers with head-stalk structures reminiscent of motor proteins, although no sequence similarities to known motor proteins were found. In physiological buffers, astrin dimers oligomerized via their globular head domains and formed aster-like structures. Silencing of astrin in HeLa cells by RNA interference resulted in growth arrest, with formation of multipolar and highly disordered spindles. Chromosomes did not congress to the spindle equator and remained dispersed. Cells depleted of astrin were normal during interphase but were unable to progress through mitosis and finally ended in apoptotic cell death. Possible functions of astrin in mitotic spindle organization are discussed.

Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.