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Aurora A is a serine/threonine kinase essential for mitotic entry and spindle assembly. Recent molecular studies have revealed the existence of multiple, distinct mechanisms of Aurora A activation, each occurring at specific subcellular locations, optimized for cellular context, and primed by signaling events including phosphorylation and oxidation., (© 2021 Tavernier et al.)

The Aurora B chromosomal passenger complex (CPC) is a conserved regulator of mitosis. Its functions require localization first to the chromosome arms and then centromeres in mitosis and subsequently the central spindle in anaphase. Here, we analyze the requirements for core CPC subunits, survivin and INCENP, and the mitotic kinesin-like protein 2 (MKLP2) in targeting to these distinct localizations. Centromere recruitment of the CPC requires interaction of survivin with histone H3 phosphorylated at threonine 3, and we provide a complete structure of this assembly. Furthermore, we show that the INCENP RRKKRR-motif is required for both centromeric localization of the CPC in metaphase and MKLP2-dependent transport in anaphase. MKLP2 and DNA bind competitively to this motif, and INCENP T59 phosphorylation acts as a switch preventing MKLP2 binding in metaphase. In anaphase, CPC binding promotes the microtubule-dependent ATPase activity of MKLP2. These results explain how centromere targeting of the CPC in mitosis is coupled to its movement to the central spindle in anaphase., (© 2020 Serena et al.)

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible "bridges"; there are no contacts with the protofilaments' curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement., (© 2019 Ng et al.)

The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity., (© 2017 Lane and Jones.)

The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning., (© 2016 Kern et al.)

The microtubule (MT) cytoskeleton bipolarizes at the onset of mitosis to form the spindle. In animal cells, the kinesin-5 Eg5 primarily drives this reorganization by actively sliding MTs apart. Its primacy during spindle assembly renders Eg5 essential for mitotic progression, demonstrated by the lethal effects of kinesin-5/Eg5 inhibitors (K5Is) administered in cell culture. However, cultured cells can acquire resistance to K5Is, indicative of alternative spindle assembly mechanisms and/or pharmacological failure. Through characterization of novel K5I-resistant cell lines, we unveil an Eg5 motility-independent spindle assembly pathway that involves both an Eg5 rigor mutant and the kinesin-12 Kif15. This pathway centers on spindle MT bundling instead of Kif15 overexpression, distinguishing it from those previously described. We further show that large populations (∼10(7) cells) of HeLa cells require Kif15 to survive K5I treatment. Overall, this study provides insight into the functional plasticity of mitotic kinesins during spindle assembly and has important implications for the development of antimitotic regimens that target this process., (© 2016 Sturgill et al.)

Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms., (© 2014 Snider et al.)

The spindle segregates chromosomes in dividing eukaryotic cells, and its assembly pathway and morphology vary across organisms and cell types. We investigated mechanisms underlying differences between meiotic spindles formed in egg extracts of two frog species. Small Xenopus tropicalis spindles resisted inhibition of two factors essential for assembly of the larger Xenopus laevis spindles: RanGTP, which functions in chromatin-driven spindle assembly, and the kinesin-5 motor Eg5, which drives antiparallel microtubule (MT) sliding. This suggested a role for the MT-associated protein TPX2 (targeting factor for Xenopus kinesin-like protein 2), which is regulated by Ran and binds Eg5. Indeed, TPX2 was threefold more abundant in X. tropicalis extracts, and elevated TPX2 levels in X. laevis extracts reduced spindle length and sensitivity to Ran and Eg5 inhibition. Higher TPX2 levels recruited Eg5 to the poles, where MT density increased. We propose that TPX2 levels modulate spindle architecture through Eg5, partitioning MTs between a tiled, antiparallel array that promotes spindle expansion and a cross-linked, parallel architecture that concentrates MTs at spindle poles., (© 2014 Helmke and Heald.)

The spindle is a dynamic self-assembling machine that coordinates mitosis. The spindle's function depends on its ability to organize microtubules into poles and maintain pole structure despite mechanical challenges and component turnover. Although we know that dynein and NuMA mediate pole formation, our understanding of the forces dynamically maintaining poles is limited: we do not know where and how quickly they act or their strength and structural impact. Using laser ablation to cut spindle microtubules, we identify a force that rapidly and robustly pulls severed microtubules and chromosomes poleward, overpowering opposing forces and repairing spindle architecture. Molecular imaging and biophysical analysis suggest that transport is powered by dynein pulling on minus ends of severed microtubules. NuMA and dynein/dynactin are specifically enriched at new minus ends within seconds, reanchoring minus ends to the spindle and delivering them to poles. This force on minus ends represents a newly uncovered chromosome transport mechanism that is independent of plus end forces at kinetochores and is well suited to robustly maintain spindle mechanical integrity., (© 2014 Elting et al.)

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN-KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression.

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.

The coordinated growth of cells and their organelles is a fundamental and poorly understood problem, with implications for processes ranging from embryonic development to oncogenesis. Recent experiments have shed light on the cell size-dependent assembly of membrane-less cytoplasmic and nucleoplasmic structures, including ribonucleoprotein (RNP) granules and other intracellular bodies. Many of these structures behave as condensed liquid-like phases of the cytoplasm/nucleoplasm. The phase transitions that appear to govern their assembly exhibit an intrinsic dependence on cell size, and may explain the size scaling reported for a number of structures. This size scaling could, in turn, play a role in cell growth and size control.

Most animal cells contain a centrosome, which comprises a pair of centrioles surrounded by an ordered pericentriolar matrix (PCM). Although the role of this organelle in organizing the mitotic spindle poles is well established, its precise contribution to cell division and cell survival remains a subject of debate. By genetically ablating key components of centriole biogenesis in chicken DT40 B cells, we generated multiple cell lines that lack centrioles. PCM components accumulated in acentriolar microtubule (MT)-organizing centers but failed to adopt a higher-order structure, as shown by three-dimensional structured illumination microscopy. Cells without centrioles exhibited both a delay in bipolar spindle assembly and a high rate of chromosomal instability. Collectively, our results expose a vital role for centrosomes in establishing a mitotic spindle geometry that facilitates correct kinetochore-MT attachments. We propose that centrosomes are essential in organisms in which rapid segregation of a large number of chromosomes needs to be attained with fidelity.

Interfaces between spindle microtubules and kinetochores were examined in diverse species by electron tomography and image analysis. Overall structures were conserved in a mammal, an alga, a nematode, and two kinds of yeasts; all lacked dense outer plates, and most kinetochore microtubule ends flared into curved protofilaments that were connected to chromatin by slender fibrils. Analyses of curvature on >8,500 protofilaments showed that all classes of spindle microtubules displayed some flaring protofilaments, including those growing in the anaphase interzone. Curved protofilaments on anaphase kinetochore microtubules were no more flared than their metaphase counterparts, but they were longer. Flaring protofilaments in budding yeasts were linked by fibrils to densities that resembled nucleosomes; these are probably the yeast kinetochores. Analogous densities in fission yeast were larger and less well-defined, but both yeasts showed ring- or partial ring-shaped structures girding their kinetochore microtubules. Flaring protofilaments linked to chromatin are well placed to exert force on chromosomes, assuring stable attachment and reliable anaphase segregation.

Accurate positioning of spindles is essential for asymmetric mitotic and meiotic cell divisions that are crucial for animal development and oocyte maturation, respectively. The predominant model for spindle positioning, termed "cortical pulling," involves attachment of the microtubule-based motor cytoplasmic dynein to the cortex, where it exerts a pulling force on microtubules that extend from the spindle poles to the cell cortex, thereby displacing the spindle. Recent studies have addressed important details of the cortical pulling mechanism and have revealed alternative mechanisms that may be used when microtubules do not extend from the spindle to the cortex.

Mitotic centromere-associated kinesin (MCAK) is a microtubule-depolymerizing kinesin-13 member that can track with polymerizing microtubule tips (hereafter referred to as tip tracking) during both interphase and mitosis. MCAK tracks with microtubule tips by binding to end-binding proteins (EBs) through the microtubule tip localization signal SKIP, which lies N terminal to MCAK's neck and motor domain. The functional significance of MCAK's tip-tracking behavior during mitosis has never been explained. In this paper, we identify and define a mitotic function specific to the microtubule tip-associated population of MCAK: negative regulation of microtubule length within the assembling bipolar spindle. This function depends on MCAK's ability to bind EBs and track with polymerizing nonkinetochore microtubule tips. Although this activity antagonizes centrosome separation during bipolarization, it ultimately benefits the dividing cell by promoting robust kinetochore attachments to the spindle microtubules.

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.

Sister chromatid cohesion provides the mechanistic basis, together with spindle microtubules, for generating tension between bioriented chromosomes in metaphase. Pericentric chromatin forms an intramolecular loop that protrudes bidirectionally from the sister chromatid axis. The centromere lies on the surface of the chromosome at the apex of each loop. The cohesin and condensin structural maintenance of chromosomes (SMC) protein complexes are concentrated within the pericentric chromatin, but whether they contribute to tension-generating mechanisms is not known. To understand how pericentric chromatin is packaged and resists tension, we map the position of cohesin (SMC3), condensin (SMC4), and pericentric LacO arrays within the spindle. Condensin lies proximal to the spindle axis and is responsible for axial compaction of pericentric chromatin. Cohesin is radially displaced from the spindle axis and confines pericentric chromatin. Pericentric cohesin and condensin contribute to spindle length regulation and dynamics in metaphase. Together with the intramolecular centromere loop, these SMC complexes constitute a molecular spring that balances spindle microtubule force in metaphase.

The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2-Bfa1 GAP complex that inhibit the mitotic exit-promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2-Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2-Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.

The mitotic exit network (MEN) is a signaling cascade that triggers inactivation of the mitotic cyclin-dependent kinases and exit from mitosis. The GTPase Tem1 localizes on the spindle pole bodies (SPBs) and initiates MEN signaling. Tem1 activity is inhibited until anaphase by Bfa1-Bub2. These proteins are also part of the spindle position checkpoint (SPOC), a surveillance mechanism that restrains mitotic exit until the spindle is correctly positioned. Here, we show that regulation of Tem1 localization is essential for the proper function of the MEN and the SPOC. We demonstrate that the dynamics of Tem1 loading onto SPBs determine the recruitment of other MEN components to this structure, and reevaluate the interdependence in the localization of Tem1, Bfa1, and Bub2. We also find that removal of Tem1 from the SPBs is critical for the SPOC to impede cell cycle progression. Finally, we demonstrate for the first time that localization of Tem1 to the SPBs is a requirement for mitotic exit.

Progenitor cells must balance self-amplification and production of differentiated progeny during development and homeostasis. In the epidermis, progenitors divide symmetrically to increase surface area and asymmetrically to promote stratification. In this study, we show that individual epidermal cells can undergo both types of division, and therefore, the balance is provided by the sum of individual cells' choices. In addition, we define two control points for determining a cell's mode of division. First is the expression of the mouse Inscuteable gene, which is sufficient to drive asymmetric cell division (ACD). However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, we show that p63, a transcriptional regulator of stratification, does not control either of these processes. These data have uncovered two important regulatory points controlling ACD in the epidermis and allow a framework for analysis of how external cues control this important choice.

The central spindle forms between segregating chromosomes during anaphase and is required for cytokinesis. Although anaphase-specific bundling and stabilization of interpolar microtubules (MTs) contribute to formation of the central spindle, it remains largely unknown how these MTs are prepared. Using live imaging of MT plus ends and an MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle occur independently of preanaphase MTs or centrosomal MT nucleation but require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during preanaphase. MTs generated in a hepatoma up-regulated protein (HURP)-dependent manner during anaphase also contribute to central spindle formation redundantly with preanaphase MTs. Based on these results, a new model for central spindle assembly is proposed.

In this issue, Pitaval et al. (2010. J. Cell Biol. doi:10.1083/jcb.201004003) demonstrate that cell geometry can regulate the elaboration of a primary cilium. Their findings and approaches are part of a historical line of inquiry investigating the role of cell shape in intracellular organization and cellular function.

Epithelial organs are made of tubes and cavities lined by a monolayer of polarized cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. The Rho guanosine triphosphatase (GTPase) Cdc42, which is a master regulator of cell polarity, regulates the formation of the central lumen in epithelial morphogenesis. However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the three-dimensional Madin-Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localizes to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects.

Coordinated cell polarization and mitotic spindle orientation are thought to be important for epithelial morphogenesis. Whether spindle orientation is indeed linked to epithelial morphogenesis and how it is controlled at the molecular level is still unknown. Here, we show that the NuMA- and Galpha-binding protein LGN is required for directing spindle orientation during cystogenesis of MDCK cells. LGN localizes to the lateral cell cortex, and is excluded from the apical cell cortex of dividing cells. Depleting LGN, preventing its cortical localization, or disrupting its interaction with endogenous NuMA or Galpha proteins all lead to spindle misorientation and abnormal cystogenesis. Moreover, artificial mistargeting of endogenous LGN to the apical membrane results in a near 90 degrees rotation of the spindle axis and profound cystogenesis defects that are dependent on cell division. The normal apical exclusion of LGN during mitosis appears to be mediated by atypical PKC. Thus, cell polarization-mediated spatial restriction of spindle orientation determinants is critical for epithelial morphogenesis.

The mechanisms that maintain the orientation of cortical polarity and asymmetric division unchanged in consecutive mitoses in Drosophila melanogaster neuroblasts (NBs) are unknown. By studying the effect of transient microtubule depolymerization and centrosome mutant conditions, we have found that such orientation memory requires both the centrosome-organized interphase aster and centrosome-independent functions. We have also found that the span of such memory is limited to the last mitosis. Furthermore, the orientation of the NB axis of polarity can be reset to any angle with respect to the surrounding tissue and is, therefore, cell autonomous.

BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mitotic timing, and spindle function, but the interdependence of these functions is unclear. We have analyzed in Drosophila melanogaster the mitotic phenotypes of kinase-dead (KD) BubR1 and BubR1 lacking the N-terminal KEN box. bubR1-KD individuals have a robust SAC but abnormal spindles with thin kinetochore fibers, suggesting that the kinase activity modulates microtubule capture and/or dynamics but is relatively dispensable for SAC function. In contrast, bubR1-KEN flies have normal spindles but no SAC. Nevertheless, mitotic timing is normal as long as Mad2 is present. Thus, the SAC, timer, and spindle functions of BubR1 are substantially separable. Timing is shorter in bubR1-KEN mad2 double mutants, yet in these flies, lacking both critical SAC components, chromosomes still segregate accurately, reconfirming that in Drosophila, reliable mitosis does not need the SAC.

Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

Sister chromatid separation at anaphase is triggered by cleavage of the cohesin subunit Scc1, which is mediated by separase. Centriole disengagement also requires separase. This dual role of separase permits concurrent control of these events for accurate metaphase to anaphase transition. Although the molecular mechanism underlying sister chromatid cohesion has been clarified, that of centriole cohesion is poorly understood. In this study, we show that Akt kinase-interacting protein 1 (Aki1) localizes to centrosomes and regulates centriole cohesion. Aki1 depletion causes formation of multipolar spindles accompanied by centriole splitting, which is separase dependent. We also show that cohesin subunits localize to centrosomes and that centrosomal Scc1 is cleaved by separase coincidentally with chromatin Scc1, suggesting a role of Scc1 as a connector of centrioles as well as sister chromatids. Interestingly, Scc1 depletion strongly induces centriole splitting. Furthermore, Aki1 interacts with cohesin in centrosomes, and this interaction is required for centriole cohesion. We demonstrate that centrosome-associated Aki1 and cohesin play pivotal roles in preventing premature cleavage in centriole cohesion.

Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein-tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.

Eukaryotic cells possess a sophisticated membrane system to facilitate diverse functions. Whereas much is known about the nature of membrane systems in interphase, the organization and function of the mitotic membrane system are less well understood. In this study, we show that epsin, an endocytic adapter protein, regulates mitotic membrane morphology and spindle integrity in HeLa cells. Using epsin that harbors point mutations in the epsin NH2-terminal homology domain and spindle assembly assays in Xenopus laevis egg extracts, we show that epsin-induced membrane curvature is required for proper spindle morphogenesis, independent of its function in endocytosis during interphase. Although several other membrane-interacting proteins, including clathrin, AP2, autosomal recessive hypercholesterolemia, and GRASP65, are implicated in the regulation of mitosis, whether they participate through regulation of membrane organization is unclear. Our study of epsin provides evidence that mitotic membrane organization influences spindle integrity.

Mitotic spindle formation and chromosome segregation depend critically on kinetochore-microtubule (KT-MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation.

Telomeric repeat binding factor 1 (TRF1) is a component of the multiprotein complex "shelterin," which organizes the telomere into a high-order structure. TRF1 knockout embryos suffer from severe growth defects without apparent telomere dysfunction, suggesting an obligatory role for TRF1 in cell cycle control. To date, the mechanism regulating the mitotic increase in TRF1 protein expression and its function in mitosis remains unclear. Here, we identify guanine nucleotide-binding protein-like 3 (GNL3L), a GTP-binding protein most similar to nucleostemin, as a novel TRF1-interacting protein in vivo. GNL3L binds TRF1 in the nucleoplasm and is capable of promoting the homodimerization and telomeric association of TRF1, preventing promyelocytic leukemia body recruitment of telomere-bound TRF1, and stabilizing TRF1 protein by inhibiting its ubiquitylation and binding to FBX4, an E3 ubiquitin ligase for TRF1. Most importantly, the TRF1 protein-stabilizing activity of GNL3L mediates the mitotic increase of TRF1 protein and promotes the metaphase-to-anaphase transition. This work reveals novel aspects of TRF1 modulation by GNL3L.

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible "wait anaphase" signal to the vicinity of the spindle.

Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.

Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a "wait anaphase" signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier-mCherry and Ndc80-green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.

Primary cilia project from the surface of most vertebrate cells and are thought to be sensory organelles. Defects in primary cilia lead to cystic kidney disease, although the ciliary mechanisms that promote and maintain normal renal function remain incompletely understood. In this work, we generated a floxed allele of the ciliary assembly gene Ift20. Deleting this gene specifically in kidney collecting duct cells prevents cilia formation and promotes rapid postnatal cystic expansion of the kidney. Dividing collecting duct cells in early stages of cyst formation fail to properly orient their mitotic spindles along the tubule, whereas nondividing cells improperly position their centrosomes. At later stages, cells lacking cilia have increased canonical Wnt signaling and increased rates of proliferation. Thus, IFT20 functions to couple extracellular events to cell proliferation and differentiation.

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.

The four-subunit Ndc80 complex, comprised of Ndc80/Nuf2 and Spc24/Spc25 dimers, directly connects kinetochores to spindle microtubules. The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules. Here, we use cryoelectron microscopy and helical image analysis to visualize the interaction of the Ndc80/Nuf2 dimer with microtubules. Our results, when combined with crystallography data, suggest that the globular domain of the Ndc80 subunit binds strongly at the interface between tubulin dimers and weakly at the adjacent intradimer interface along the protofilament axis. Such a binding mode, in which the Ndc80 complex interacts with sequential alpha/beta-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules. Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends.

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.

Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.

The condensin complex has a fundamental role in chromosome dynamics. In this study, we report that accumulation of Schizosaccharomyces pombe condensin at mitotic kinetochores and ribosomal DNAs (rDNAs) occurs in multiple steps and is necessary for normal segregation of the sister kinetochores and rDNAs. Nuclear entry of condensin at the onset of mitosis requires Cut15/importin alpha and Cdc2 phosphorylation. Ark1/aurora and Cut17/Bir1/survivin are needed to dock the condensin at both the kinetochores and rDNAs. Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites. Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs. The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells.

Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.

To investigate the dynamics of centromere organization, we have assessed the exchange rates of inner centromere proteins (CENPs) by quantitative microscopy throughout the cell cycle in human cells. CENP-A and CENP-I are stable centromere components that are incorporated into centromeres via a "loading-only" mechanism in G1 and S phase, respectively. A subfraction of CENP-H also stays stably bound to centromeres. In contrast, CENP-B, CENP-C, and some CENP-H and hMis12 exhibit distinct and cell cycle-specific centromere binding stabilities, with residence times ranging from seconds to hours. CENP-C and CENP-H are immobilized at centromeres specifically during replication. In mitosis, all inner CENPs become completely immobilized. CENPs are highly mobile throughout bulk chromatin, which is consistent with a binding-diffusion behavior as the mechanism to scan for vacant high-affinity binding sites at centromeres. Our data reveal a wide range of cell cycle-specific assembly plasticity of the centromere that provides both stability through sustained binding of some components and flexibility through dynamic exchange of other components.

Production of Ran-guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)-containing proteins from Xenopus laevis egg extracts. This NLS protein fraction contains the MT stabilization activity. After further purification, we used mass spectrometry to identify proteins in active fractions, including cyclin-dependent kinase 11 (Cdk11). Cdk11 localizes on spindle poles and MTs in Xenopus culture cells and egg extracts. Recombinant Cdk11 demonstrates RanGTP-dependent MT stabilization activity, whereas a kinase-dead mutant does not. Inactivation of Cdk11 in egg extracts blocks RanGTP-dependent MT stabilization and dramatically decreases the spindle assembly rate. Simultaneous depletion of TPX2 completely inhibits centrosome-dependent spindle assembly. Our results indicate that Cdk11 is responsible for RanGTP-dependent MT stabilization around chromosomes and that this local stabilization is essential for normal rates of spindle assembly and spindle function.

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating gamma-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires gamma-tubulin and microtubule assembly. These results indicate that gamma-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.