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Huang et al. reveal that the microcephaly-related protein WDR62 possesses an intrinsic affinity for curved microtubules and regulates spindle dynamics by functioning as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin., WDR62 is a microcephaly-related, microtubule (MT)-associated protein (MAP) that localizes to the spindle pole and regulates spindle organization, but the underlying mechanisms remain elusive. Here, we show that WDR62 regulates spindle dynamics by recruiting katanin to the spindle pole and further reveal a TPX2–Aurora A–WDR62–katanin axis in cells. By combining cellular and in vitro experiments, we demonstrate that WDR62 shows preference for curved segments of dynamic GDP-MTs, as well as GMPCPP- and paclitaxel-stabilized MTs, suggesting that it recognizes extended MT lattice. Consistent with this property, WDR62 alone is inefficient in recruiting katanin to GDP-MTs, while WDR62 complexed with TPX2/Aurora A can potently promote katanin-mediated severing of GDP-MTs in vitro. In addition, the MT-binding affinity of WDR62 is autoinhibited through JNK phosphorylation-induced intramolecular interaction. We propose that WDR62 is an atypical MAP and functions as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin to orchestrate the regulation of spindle dynamics.

Kornakov et al. investigate mechanisms of bipolar spindle assembly, a critical prerequisite for accurate chromosome segregation in mitosis. They define the molecular organization of an interaction interface between the conserved microtubule plus-end tracking protein EB1 and the minus-end motor Kinesin-14 and demonstrate with specific mutations that this complex is required for proper organization of the metaphase spindle., Kinesin-14s are conserved molecular motors required for high-fidelity chromosome segregation, but their specific contributions to spindle function have not been fully defined. Here, we show that key functions of budding yeast Kinesin-14 Cik1-Kar3 are accomplished in a complex with Bim1 (yeast EB1). Genetic complementation of mitotic phenotypes identifies a novel KLTF peptide motif in the Cik1 N-terminus. We show that this motif is one element of a tripartite binding interface required to form a high-affinity Bim1–Cik1-Kar3 complex. Lack of Bim1-binding by Cik1-Kar3 delays cells in mitosis and impairs microtubule bundle organization and dynamics. Conversely, constitutive targeting of Cik1-Kar3 to microtubule plus ends induces the formation of nuclear microtubule bundles. Cells lacking the Bim1–Cik1-Kar3 complex rely on the conserved microtubule bundler Ase1/PRC1 for metaphase spindle organization, and simultaneous loss of plus-end targeted Kar3 and Ase1 is lethal. Our results reveal the contributions of an EB1–Kinesin-14 complex for spindle formation as a prerequisite for efficient kinetochore clustering and bi-orientation.

Gradients of Aurora A and B kinase activity at the mitotic spindle are crucial for chromosome alignment. Poser et al. show how Aurora A at the spindle poles promotes chromosome congression by activating the chromokinesin KIF4A., Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase, thereby locally regulating factors that promote spindle organization, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the central spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase central spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1 or are deficient for Aurora kinase regulation. By analyzing these mutants, we show that Aurora A phosphorylates the condensin I–dependent pool of KIF4A and thus actively promotes chromosome congression from the spindle poles to the metaphase plate.

Cytoplasmic dynein is a large minus end–directed motor complex with multiple functions during cell division. The dynein complex interacts with various adaptor proteins, including the dynactin complex, thought to be critical for most dynein functions. Specific activities have been linked to several subunits and adaptors, but the function of the majority of components has remained elusive. Here, we systematically address the function of each dynein–dynactin subunit and adaptor protein in mitosis. We identify the essential components that are required for all mitotic functions of dynein. Moreover, we find specific dynein recruitment factors, and adaptors, like Nde1/L1, required for activation, but largely dispensable for dynein localization. Most surprisingly, our data show that dynactin is not required for dynein-dependent spindle organization, but acts as a dynein recruitment factor. These results provide a comprehensive overview of the role of dynein subunits and adaptors in mitosis and reveal that dynein forms distinct complexes requiring specific recruiters and activators to promote orderly progression through mitosis.

Spatially dispersed nucleation and minus end–directed transport of microtubule end disassembly activity can lead to bipolar spindle assembly., The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5–like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.

RCC1 is the only known exchange factor for the Ran guanosine triphosphatase and performs essential roles in nuclear transport, spindle organization, and nuclear envelope formation. RCC1 binds to chromatin through a bimodal attachment to DNA and histones, and defects in binding cause chromosome missegregation. Chromatin binding is enhanced by apo-Ran. However, the mechanism underlying this regulation has been unclear. We now demonstrate that the N-terminal tail of RCC1 is essential for association with DNA but inhibits histone binding. Apo-Ran significantly promotes RCC1 binding to both DNA and histones, and these effects are tail mediated. Using a fluorescence resonance energy transfer biosensor, we detect conformational changes in the tail of RCC1 coupled to the two binding modes and in response to interactions with Ran and importin-α. The biosensor also reports changes accompanying mitosis in living cells. We propose that Ran induces an allosteric conformational switch in the tail that exposes the histone-binding surface on RCC1 and facilitates association of the positively charged tail with DNA.

Centrosomes and microtubules play crucial roles during cell division and differentiation. Spermatogenesis is a useful system for studying centrosomal function since it involves both mitosis and meiosis, and also transformation of the centriole into the sperm basal body. Centrosomin is a protein localized to the mitotic centrosomes in Drosophila melanogaster. We have found a novel isoform of centrosomin expressed during spermatogenesis. Additionally, an anticentrosomin antibody labels both the mitotic and meiotic centrosomes as well as the basal body. Mutational analysis shows that centrosomin is required for spindle organization during meiosis and for organization of the sperm axoneme. These results suggest that centrosomin is a necessary component of the meiotic centrosomes and the spermatid basal body.

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.