Understanding the regulation of the submembranous actomyosin cortex of adherent cells by micro-mechanical environment

Kumar, Rinku (2021) Understanding the regulation of the submembranous actomyosin cortex of adherent cells by micro-mechanical environment. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Rinku Kumar (12RS030)) 12RS030.pdf - Submitted Version Restricted to Repository staff only Download (7MB)

Abstract

The precisely controlled cell shape change is at the heart of many biological processes such as cell division, cell migration and tissue morphogenesis. A thin submicron layer of actomyosin network lies beneath the plasma membrane called actin cortex or cortical actin, is responsible for driving these cellular deformations. The cell undergoes local modulation in cortex mechanics to facilitate cell deformation with the help of molecular regulators. Therefore, these molecular regulators determine cortex mechanics such as tension and viscosity by precise changes in the organization of the actomyosin network in space and time. Thus, understanding cell deformation requires knowledge of cortical network organization. While study of the cortical network organization in cells at the nano-metric scale poses various challenges, cortex thickness has been proposed to reflect mechanical state of the cortex. Cortex thickness is believed to be regulated by filament length regulator, myosin motor activity and actin dynamics. Despite these insights, it was largely unexplored how the external micromechanical environment such as cell contact area, substrate stiffness, in harmony with the internal regulators controls cortex thickness. In this thesis work, we have demonstrated that cortex thickness is not only regulated by cell spread area and cell traction force – but these parameters also decide the role myosin IIA plays in its regulation. We report cell with high spread area (> 450 μm²) shows thinner cortex, well-separated layers, and sensitive to myosin IIa activity and gel stiffness (traction force). However, low spread cells (< 400 μm²) display relatively thicker (thicker than high spread cell) cortex, insensitive to myosin IIa activity and gel stiffness. Low spread cell has limited myosin IIA mechanosensing due to its fast turnover and lower actin bound fraction in the cortex. Cofilin reported as a competitive inhibitor of myosin IIA does overexpress in these cells. Besides these, restricting spread area also reduces cell volume, surface area and traction forces and latter displays a stronger correlation to the thickness of sub-membranous actin layer. The volume reduction is endocytosis dependent and not essential for the thickening of the actin layer. To understand the causal relationship between cortex thickness, spread area and filaments organization; we induced gradual spread area reduction and found enhanced actin filaments fragmentation, enhanced actin dynamics together with gradual increase and oscillation of cortex thickness. Cortex thickening by spread area reduction also holds true on differentiating monocytes to macrophage. To understand how traction forces, that act at the basal plane and mediated by stress fibres – affect the cortical actin network and vice versa, we first induce an increase in traction forces by cell stretching. Stretching enhances cortex thickness – in line with expectations from the probable fragmentation of stress fibres and amplified active contraction. Finally, we enhance or weaken the binding of the cortex to the membrane by inhibiting or activating Ezrin proteins and report that traction forces are reduced on activation while decreased on inhibition of Ezrin proteins. Additionally, we also find enhanced ezrin in conditions that thicken the cortex. This points to the fact that the bonds mediated by Ezrin between the cortex and the membrane reduces the alignment of cortex-contractility with that of the stress fibres. Therefore, Ezrin also acts as an important factor controlling the cortex architecture and mechanics.

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