Julie C. Canman

Rho GTPases in animal cell cytokinesis: An occupation by the one percent

Rho GTPases are molecular switches that elicit distinct effects on the actomyosin cytoskeleton to accurately promote cytokinesis. Although they represent less than 1% of the human genome, Rho GTPases exert disproportionate control over cell division. Crucial to this master regulatory role is their localized occupation of specific domains of the cell to ensure the assembly of a contractile ring at the proper time and place. RhoA occupies the division plane and is the central positive Rho family regulator of cytokinesis. Rac1 is a negative regulator of cytokinesis and is inactivated within the division plane while active Rac1 occupies the cell poles. Cdc42 regulation during cytokinesis is less studied, but thus far a clear role has only been shown during polar body emission. Here we review what is known about the function of Rho family GTPases during cell division, as well as their upstream regulators and known downstream cytokinetic effectors.

High-Resolution Temporal Analysis Reveals a Functional Timeline for the Molecular Regulation of Cytokinesis

To take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ~17 s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the Caenorhabditis elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, although its GAP activity is only required until just prior to closure. Finally, the chromosomal passenger complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation.

Kinetochore components are required for central spindle assembly

A critical structure poised to coordinate chromosome segregation with division plane specification is the central spindle that forms between separating chromosomes after anaphase onset. The central spindle acts as a signalling centre that concentrates proteins essential for division plane specification and contractile ring constriction. However, the molecular mechanisms that control the initial stages of central spindle assembly remain elusive. Using Caenorhabditis elegans zygotes, we found that the microtubule-bundling protein SPD-1PRC1 and the motor ZEN-4MKLP-1 are required for proper central spindle structure during its elongation. In contrast, we found that the kinetochore controls the initiation of central spindle assembly. Specifically, central spindle microtubule assembly is dependent on kinetochore recruitment of the scaffold protein KNL-1, as well as downstream partners BUB-1, HCP-1/2CENP-F and CLS-2CLASP; and is negatively regulated by kinetochore-associated protein phosphatase 1 activity. This in turn promotes central spindle localization of CLS-2CLASP and initial central spindle microtubule assembly through its microtubule polymerase activity. Together, our results reveal an unexpected role for a conserved kinetochore protein network in coupling two critical events of cell division: chromosome segregation and cytokinesis.

Cortical PAR polarity proteins promote robust cytokinesis during asymmetric cell division

In asymmetrically dividing C. elegans embryos, the core cortical PAR proteins are required to retain septin and anillin at the anterior cortex away from the contractile ring and to promote normal F-actin levels at the contractile ring and successful cytokinesis.

Stuck in the middle: Rac, adhesion, and cytokinesis.

Rho family small GTPases (Rac, RhoA, and Cdc42) function at the core of cytokinesis, the physical division of one cell into two. In this issue, Bastos et al. (2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201204107) identify a new role for Rac inhibition: to release cell adhesion at the division plane and allow efficient constriction of the contractile ring. They show that the GTPase-activating protein, CYK4, suppresses equatorial cell substrate adhesion by inhibiting Rac and therefore its effectors ARFGEF7 and PAK1/2.

A Drosophila model of Fragile X syndrome exhibits defects in phagocytosis by innate immune cells

Fragile X syndrome, the most common known monogenic cause of autism, results from the loss of FMR1, a conserved, ubiquitously expressed RNA-binding protein. Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. We found that Drosophila melanogaster Fmr1 mutants exhibit increased sensitivity to bacterial infection and decreased phagocytosis of bacteria by systemic immune cells. Using tissue-specific RNAi-mediated knockdown, we showed that Fmr1 plays a cell-autonomous role in the phagocytosis of bacteria. Fmr1 mutants also exhibit delays in two processes that require phagocytosis by glial cells, the immune cells in the brain: neuronal clearance after injury in adults and the development of the mushroom body, a brain structure required for learning and memory. Delayed neuronal clearance is associated with reduced recruitment of activated glia to the site of injury. These results suggest a previously unrecognized role for Fmr1 in regulating the activation of phagocytic immune cells both in the body and the brain.

Cytokinesis: thinking outside the cell.

How might the extracellular matrix contribute to cytokinesis? In a recent report, evidence is presented that the conserved extracellular matrix protein hemicentin(HIM-4) is required for cytokinesis in worms and mice.

Chromosome segregation occurs by microtubule pushing in oocytes

During cell division, spindle microtubules ensure an equal repartition of chromosomes between the two daughter cells. While the kinetochore-dependent mechanisms that drive mitotic chromosome segregation are well understood, in oocytes of most species atypical spindles assembled in absence of centrosomes entail poorly understood mechanisms of chromosome segregation. In particular, the structure(s) responsible for force generation during meiotic chromosome separation in oocytes is unclear. Using quantitative light microscopy, electron tomography, laser-mediated ablation, and genetic perturbations in the Caenorhabditis elegans oocyte, we studied the mechanism of chromosome segregation in meiosis. We find spindle poles are largely dispensable, and in fact act as brakes for chromosome segregation. Instead, our results suggest that CLS-2-dependent microtubules of the meiotic central spindle, located between the segregating chromosomes and aligned along the axis of segregation, are essential. Our results support a model in which inter-chromosomal microtubules of the central spindle push chromosomes apart during meiotic anaphase in oocytes.In oocytes of most species atypical spindles assembled in the absence of centrosomes drive chromosome segregation, however the forces driving this process are unclear. Here the authors found that spindle poles are largely dispensable and that inter-chromosomal microtubules of the central spindle control chromosomal segregation.

Using fast-acting temperature-sensitive mutants to study cell division in Caenorhabditis elegans.

Fast-acting temperature-sensitive (ts) mutations are powerful conditional tools for studying transient cellular processes such as cytokinesis. Fast-acting ts cytokinesis-defective mutants are functional at the permissive temperature; yet show a fully penetrant loss-of-function cytokinesis failure phenotype when upshifted to the restrictive temperature. Fast-acting ts mutations thus allow functional tunability and rapid and reversible protein inactivation by simply shifting the temperature at precise times throughout cell division. In this chapter, we describe several techniques and discuss various approaches for harnessing the power of fast-acting ts mutants to study cytokinesis in Caenorhabditis elegans using both simple passive heat transfer and more advanced fluidic-based thermal control systems. We also provide detailed protocols for standard dissection, mounting, and imaging of early worm embryos.

CYK-4 regulates Rac, but not Rho, during cytokinesis

The roles of the Rho-family GAP CYK-4 and small GTPase Rac during cytokinesis are examined in Caenorhabditis elegans embryos. CYK-4 opposes Rac (and potentially Cdc42) activity during cytokinesis. There is no evidence that CYK-4 is upstream of Rho activity or that Rac disruption is a general suppressor of cytokinesis failure.