Identifying Cues that Regulate the Position of the Contractile Ring during Cytokinesis

Cytokinesis is the process where a mother cell cleaves into two daughter cells and is driven by the constriction of an actomyosin ring. Formation and ingression of the contractile ring is regulated by the mitotic spindle to couple cytokinesis with the segregation of sister chromatids. The central spindle forms in anaphase and recruits Ect2, a GEF that activates RhoA, for F-actin polymerization and nonmuscle myosin activation to assemble the ring in the equatorial plane. However, the molecular mechanism that regulates the localization of contractile proteins is poorly understood. For example, astral microtubules exclude the localization of contractile proteins at the polar cortex, but the molecular pathway is not known. Furthermore, other cues likely regulate the localization of contractile proteins at the polar cortex. In this study, I investigated the role of chromatin in mediating the polar exclusion of contractile proteins in Hela cells. First, I measured the minimum distance between chromatin and the boundary of accumulated contractile proteins during cytokinesis in control cells, and in cells treated with different conditions that affect the central spindle and/or astral microtubules. I found that chromatin position closely correlates with the localization of contractile proteins within 3-4 µm of the cortex. This suggests that chromatin has a diffusible cue that can regulate the organization of actomyosin. I also found that over-expression of active Ran targeted to the equatorial membrane alters anillin localization and causes asymmetric furrow ingression or oscillation. In addition, inactivating Ran-GTP results in the global localization of myosin and cytokinesis phenotypes. My data supports the idea that chromatin, likely via Ran-GTP, works in combination with the central spindle and astral microtubules to ensure that the contractile ring forms at the correct time and location during cytokinesis

Interaction of microtubules and actin during the post-fusion phase of exocytosis

Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis