Matthew S. Savoian

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 orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression

We address the relative roles of astral and central spindle microtubules (MTs) in cytokinesis of Drosophila melanogaster primary spermatocytes. Time-lapse imaging studies reveal that the central spindle is comprised of two MT populations, “interior” central spindle MTs found within the spindle envelope and “peripheral” astral MTs that probe the cytoplasm and initiate cleavage furrows where they contact the cortex and form overlapping bundles. The MT-associated protein Orbit/Mast/CLASP concentrates on interior rather than peripheral central spindle MTs. Interior MTs are preferentially affected in hypomorphic orbit mutants, and consequently the interior central spindle fails to form or is unstable. In contrast, peripheral MTs still probe the cortex and form regions of overlap that recruit the Pav-KLP motor and Aurora B kinase. orbit mutants have disorganized or incomplete anillin and actin rings, and although cleavage furrows initiate, they ultimately regress. Our work identifies a new function for Orbit/Mast/CLASP and identifies a novel MT population involved in cleavage furrow initiation.

The rate of poleward chromosome motion is attenuated in Drosophila zw10 and rod mutants.

Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuated throughout the division process, and that chromosome disjunction at anaphase is highly asynchronous. Our results show that ZW10 protein, together with Rod, is involved in production and/or regulation of the force reponsible for poleward chromosome motion.

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.

The 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.

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.

Drosophila Klp67A is required for proper chromosome congression and segregation during meiosis I.

Drosophila Klp67A belongs to the Kip3 subfamily of Kinesin-type microtubule catastrophe factors. In primary spermatocytes, loss of klp67A leads to defects in karyokinesis and cytokinesis. We show that these cells formed disorganised, bipolar spindles that contained increased numbers of microtubules. The kinetochore fibres were wavy and bent, whereas astral microtubules appeared abnormally robust and formed cortical bundles. Time-lapse studies revealed that during biorientation, the chromosomes in klp67A mutant cells continued to reorient for about twice as long as those in control cells. Metaphase plates were poorly defined in the mutants and often formed at non-equatorial positions. Consistent with the above abnormalities in chromosome congression, we found that in wild-type cells Klp67A associated with prometaphase/metaphase kinetochores before redistributing to the central spindle at anaphase onset. Although the timing of this redistribution of kinetochores argues against a role in anaphase chromosome segregation, dyads in the mutants disjoined but exhibited greatly diminished poleward velocities. They travelled on average at approximately 34% of the velocity of their wild-type counterparts and often decondensed at non-polar locations. Hypomorphic mutations of klp67A may lead to segregation defects.

Klp67A destabilises pre-anaphase microtubules but subsequently is required to stabilise the central spindle

Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which results in abnormally long and stable pre-anaphase microtubules. Here we examine its role during cytokinesis in Drosophila primary spermatocytes that require the coordinated interaction of an interior and peripheral set of central spindle microtubules. In mutants anaphase B spindles elongated with normal kinetics but bent towards the cortex. Both peripheral and interior spindle microtubules then formed diminished bundles of abnormally positioned central spindle microtubules associated with the pavarotti-KLP and KLP3A motor proteins. The minus ends of these were poorly aligned as revealed by Asp protein localisation. Furrows always initiated at the sites of central spindle bundles but could be unilateral or nonequatorially positioned. Ectopic furrows were stimulated by the interior central spindle and formed only after this structure buckled and contacted the cortex. Furrows often halted and regressed as they could not be sustained by the central spindles that became increasing unstable over time and often completely degraded. Consistent with this, actin and anillin failed to form homogenous bands. Thus, the Klp67A microtubule catastrophe factor is required for cytokinesis by regulating both the formation and stability of the central spindle.

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.

Centromeric binding and activity of Protein Phosphatase 4

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis.