Elucidating novel regulators of cytokinesis

Mangal, Sriyash (2020): Elucidating novel regulators of cytokinesis. Dissertation, LMU München: Fakultät für Biologie

Abstract

Cytokinesis is the final event of cell division in which the mother cell splits into two daughter cells. During cytokinesis, the contractile ring is carefully positioned between the separating chromosomes by anaphase spindle. While the spindle midzone, located between segregating chromosomes, promotes the accumulation of contractile ring components at the equator, the centrosomal microtubule asters prevent the accumulation of contractile ring proteins at the cell poles. Despite rigorous research, the identity of aster derived inhibitory molecules(s) remains elusive and how cytokinesis regulators like Ect2 and RhoA are activated in a narrow equatorial zone is not properly understood. To identify novel regulators of this signalling pathway, high-throughput RNAi screen was performed in HeLa cells and cortical localization of contractile ring component GFP-tagged anillin was analyzed manually. In total, 7553 genes comprising druggable human genome were screened and 18 new genes were identified to play a role in regulating anillin localization at cell poles or equator. Among the 18 new candidate genes, Protein Kinase N2 (PKN2) and Septin 7 were the two most exciting candidates as they directly interact with RhoA and anillin, respectively and were further characterized. PKN2, a known RhoA effector, inhibited anillin localization on the cell poles whereas contractile ring component Septin 7 promoted anillin localization at the cell equator. Remarkably, the role of PKN2 and Septin7 in regulating anillin location during anaphase was found to be conserved in C. elegans one-cell embryos. It was previously shown that TPXL-1 mediated activation of Aurora A during anaphase is required for clearing anillin from the anterior pole in C. elegans one-cell embryos. To investigate whether Aurora A plays a similar role in clearing other contractile ring proteins from the anterior pole, localization of f-actin was analysed in TPXL-1 depleted embryos using a f-actin binding probe LifeAct fused to mKate2. Similar to anillin, TPXL-1 was found to be involved in clearing f-actin from the anterior pole during anaphase and the clearing defect was confirmed not to be a consequence of altered microtubule dynamics. Moreover, ectopic localization of Aurora A at the cell cortex induced by inhibiting PP6, a phosphatase that negatively regulates Aurora A activation, led to a significant reduction in anillin localization at cell equator and poles. Consistent with the observations in C. elegans, inhibition of Aurora A in HeLa cells by small molecular inhibitor MK-5108 resulted in increased accumulation of anillin on the polar cortex and a wider anillin zone at cell equator. Based on these findings, it is proposed that TPXL-1 activates Aurora A on the microtubule asters which diffuses to the adjacent cell poles and inhibits localization of contractile ring proteins. Finally, a rapamycin-inducible dimerization system was established in C. elegans using FRB and FKBP-12 domains of mTOR signaling pathways. In future, this protein dimerization tool can be used to target Aurora A and TPXL-1 to the plasma membrane and determine whether ectopic localization of TPXL-1 and Aurora A, without inhibiting PP6, can result in anillin localization defects. In summary, high-throughput RNAi screen revealed 18 new regulators of cytokinesis, two of which (Sept7 and PKN2) were further validated in HeLa cells and C. elegans one-cell embryos. In addition, Aurora A was shown to restrict the localization of contractile ring proteins to the cell equators in both the model systems.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Cytokinesis, TPXL-1, Aurora A, RNAi screen, protein dimerization in C. elegans, PKN2, Septin 7, anaphase cortical patterning
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 5. Juni 2020
• 1. Bericht­erstatter:in: Zanin, Esther