[
International Worm Meeting,
2021]
Cytokinesis occurs through the ingression of a contractile ring that cleaves the daughter cells. This process is tightly controlled to prevent fate changes or aneuploidy, and the core cytokinesis machinery is highly conserved among metazoans. The central dogma is that spindle-dependent pathways regulate ring assembly, but studies have shown that spindle-independent pathways do so as well. In human cells, we found that active Ran is a chromatin-associated cue that forms an inverse gradient with importins that can control protein function near the cortex. We found that importin-binding is required for the cortical localization and function of anillin, a ring scaffold. To study how requirements for different cytokinesis pathways vary with cell type, we studied cytokinesis in the differently fated AB and P1 cells of the early embryo. We found that AB and P1 cells have different assembly kinetics supported by different levels and cortical patterning of equatorial non-muscle myosin II (NMY-2). The ring assembles rapidly in AB cells, which has higher midplane levels and cortical patches of NMY-2. In contrast, the levels of NMY-2 are lower and there are no/few patches in P1 cells, which have slower ring assembly. Depleting polarity regulators (PAR-1, -3 or -6) equalized assembly kinetics, indicating that kinetics depend on cell fate. By generating stable tetraploid strains, we show that differences in kinetics also depend on cell size. Diploid AB-sized tetraploid P1 cells had faster ring assembly supported by higher levels of NMY-2. However, ring assembly was slower in tetraploid vs. diploid AB cells, which had very high levels of NMY-2. This suggests that ideal NMY-2 levels support ring assembly, which is hindered when levels are too low or high, and that P1 cells operate close to a minimum threshold. Next, we determined if the chromatin pathway controls differences in AB and P1 cell ring assembly. We found that depleting the RanGEF (RAN-3/RCC1) caused ring assembly to occur equally and rapidly in both AB and P1 cells, which may be due to increased importin-regulation of cortical proteins. In support of this, the faster kinetics were suppressed by co-depletion of ECT-2 (RhoGEF). Interestingly, ANI-1 (anillin) suppressed kinetics in AB cells but not P1, suggesting that the chromatin pathway functions differently in these cell types. Collectively, our results show that there are different pathway requirements in AB and P1 cells that supports differences in cytokinesis.
[
International Worm Meeting,
2017]
Cytokinesis physically divides a cell into two daughters at the end of mitosis and must be highly robust to avoid aneuploidy and cell fate changes. A RhoA-dependent actomyosin contractile ring ingresses to divide the appropriate genetic and cell fate determinants into each daughter. The prevailing dogma in the field is that the mitotic spindle regulates contractile ring assembly and ingression. However, microtubule-independent mechanisms also regulate cytokinesis, and may be crucial in polarized cells. During metaphase, a Ran-GTP gradient forms around chromatin where it mediates mitotic spindle assembly by releasing importins a/ beta complexes from microtubule-binding proteins and motors. Our data suggests that the Ran-GTP gradient persists in early anaphase where it affects the localization of contractile proteins for cytokinesis in human cells. Importantly, one of the Ran targets may be anillin, which is a scaffold that binds to and coordinates actin, myosin, RhoA and its upstream regulators, microtubules, and phospholipids. We found that human anillin contains a C-terminal nuclear localization signal (NLS) that binds to importin- beta . Point mutations in the NLS delay anillin's recruitment to the cortex, and the contractile ring oscillates and subsequently fails to ingress in a subset of cells rescued with this mutant. Interestingly, mutant anillin's localization is strongly demarcated by astral microtubules, suggesting that the mitotic spindle dominantly regulates cytokinesis in these cells. We hypothesize that the Ran-GTP regulation of cytokinesis is conserved among metazoans, and the strength of this pathway is proportional to ploidy, cell geometry and/or cell volume. To test this, we are determining if the Ran pathway regulates cytokinesis in early C. elegans embryos. Studies showed that there is a negative correlation between the rate of furrow ingression and cell volume as cells decrease in size through the first four rounds of divisions (Carvalho et al., 2009). We are determining how perturbing the Ran pathway alters the rate of furrow ingression and the localization of contractile proteins during these divisions. Also, we are determining if ANI-1 (C. elegans anillin), which is highly conserved with human anillin, contains an NLS that may function in the same way as human anillin. We also will determine if the Ran pathway regulates other well-known cytokinesis components. Carvalho et al. (2009) Cell 137: 926-37.