Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells

Drosophila Klp67A binds prophase kinetochores to subsequently regulate congression and spindle length

The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite their highly conserved roles in chromosome alignment and spindle dynamics, remain poorly characterised. Here, we report that the Drosophila kinesin-8 protein, Klp67A, exists in two spatially and functionally separable metaphase pools: at kinetochores and along the spindle. Fixed and live-cell analyses of different Klp67A recombinant variants indicate that this kinesin-8 first collects at kinetochores during prophase and, by metaphase, localises to the kinetochore outerplate. Although the catalytic motor activity of Klp67A is required for efficient kinetochore recruitment at all times, microtubules are entirely dispensable for this process. The tail of Klp67A does not play a role in kinetochore accumulation, but is both necessary and sufficient for spindle association. Using functional assays, we reveal that chromosome position and spindle length are determined by the microtubule-depolymerising motor activity of Klp67A exclusively when located at kinetochores, but not along the spindle. These data reveal that, unlike other metazoan kinesin-8 proteins, Klp67A binds the nascent prophase and mature metaphase kinetochore. From this location, Klp67A uses its motor activity to ensure chromosome alignment and proper spindle length

Drosophila Klp67A binds prophase kinetochores to subsequently regulate congression and spindle length

The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite their highly conserved roles in chromosome alignment and spindle dynamics, remain poorly characterised. Here, we report that the Drosophila kinesin-8 protein, Klp67A, exists in two spatially and functionally separable metaphase pools: at kinetochores and along the spindle. Fixed and live-cell analyses of different Klp67A recombinant variants indicate that this kinesin-8 first collects at kinetochores during prophase and, by metaphase, localises to the kinetochore outerplate. Although the catalytic motor activity of Klp67A is required for efficient kinetochore recruitment at all times, microtubules are entirely dispensable for this process. The tail of Klp67A does not play a role in kinetochore accumulation, but is both necessary and sufficient for spindle association. Using functional assays, we reveal that chromosome position and spindle length are determined by the microtubule-depolymerising motor activity of Klp67A exclusively when located at kinetochores, but not along the spindle. These data reveal that, unlike other metazoan kinesin-8 proteins, Klp67A binds the nascent prophase and mature metaphase kinetochore. From this location, Klp67A uses its motor activity to ensure chromosome alignment and proper spindle length

Cleavage furrow formation and ingression during animal cytokinesis: a microtubule legacy

Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independent daughter cells at the end of cell division. Although the metazoan mitotic spindle has been implicated in the placement and advancement of the cleavage furrow, the molecules responsible for these processes have remained elusive. Recent studies have provided insights into the role of different microtubule structures and associated proteins in cleavage furrow positioning and ingression together with the signalling events that regulate the dynamics of the equatorial cell cortex during cytokinesis. We try to unify these findings into a general model of cytokinesis in which both astral and central spindle microtubules have the ability to induce furrowing. We further propose that the evolutionarily conserved centralspindlin complex serves as a master controller of cell cleavage in Drosophila by promoting both furrow formation and ingression. The same mechanism might be conserved in other organisms

Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac

The contractile ring is a highly dynamic structure, but how this dynamism is accomplished remains unclear. Here, we report the identification and analysis of a novel Drosophila gene, sticky (sti), essential for cytokinesis in all fly proliferating tissues. sti encodes the Drosophila orthologue of the mammalian Citron kinase. RNA interference–mediated silencing of sti in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such STICKY-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes suggesting that these GTPases antagonistically regulate STICKY functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. We discuss that antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis

Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo

The metaphase-spindle steady-state length occurs as spindle microtubules `flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance

Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo

The metaphase-spindle steady-state length occurs as spindle microtubules `flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance

RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis

Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, time-lapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAP-signaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site

PITSLRE/CDK11ᵖ⁵⁸ protein kinase promotes centrosome maturation and bipolar spindle formation

The CDK11 (cyclin‐dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110‐kDa isoform is expressed at constant levels during the cell cycle and the shorter 58‐kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. γ‐Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11ᵖ⁵⁸ isoform, but not the CDK11ᵖ¹¹⁰ isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11ᵖ⁵⁸ in centrosome maturation and bipolar spindle morphogenesis