Bovellan, MK; (2012) Assembly and composition of the cellular actin cortex. Doctoral thesis , UCL (University College London).

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Abstract

The actin cortex is a thin layer of F-actin, actin regulating proteins, and myosin under the cell membrane. It has a fundamental role in cell morphogenesis, motility, mitosis, cytokinesis, and cell shape maintenance. Yet, how the cell cortex forms as well as the protein composition of the cortex remain unknown. In this study using M2 melanoma cell blebs and metaphase HeLa cells I describe proteins needed for cortex reassembly and reveal the protein composition of the actin cortex. Blebs represent a natural situation in which the membrane is transiently devoid of an actin cortex. Thus the formation of a new actin cortex in the bleb can be studied. Blebs also provide a cell fraction enriched in F-actin cortex, making them an ideal model to study cortex proteomics. After having shown that no nucleation independent mechanism participate in cortex reassembly in membrane blebs, I discovered by localisation and silencing studies, that formin Diaph1 and the Arp2/3 complex are needed for the cortex nucleation, as both localised to the cortex and depletion of either led to cortical defects. Simultaneous depletion of Diaph1 and the Arp2/3 complex led to disappearance of the cellular cortex, suggesting that together they provide the majority of the F-actin in the cortex. Cortex composition studies of blebs isolated from blebbing cells revealed the presence of many actin binding proteins at the cortex. Localisation and depletion studies revealed that capping, and effective actin turnover are essential for cortical stability and that flightless-I may regulate cortex nucleation together with Rho GTPases. These findings have important implications for our understanding of cortical properties, which are not only important for cell morphogenesis and shape maintenance, but facilitate tumour growth and dissemination.

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