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Analysis of cortical actin dynamics and its regulatory proteins in living cells

Zhang, Yanshu 京都大学 DOI:10.14989/doctor.k23334

2021.03.23

概要

Intracellular actin-based architectures play critical roles in various cellular functions and events, such as cell migration, cell division, mechanical response and tissue organization. Cell cortex is composed of a random meshwork of actin filaments and other actin-related proteins, and lies beneath the plasma membrane. Due to technical limitations, the molecular mechanism maintaining and regulating the cortical actin dynamics has been less understood than other actin-based architectures such as stress fibers. In this study, I tried to establish a microscope technique to visualize and analyze the dynamics of individual actin filaments in a living cell cortex by utilizing high-speed atomic force microscope (HS-AFM), and applied it for functional analyses of actin-regulating proteins. I used a tip-scan type HS-AFM to visualize actin filaments in the cell cortex by applying slightly deeper indentation of the probe into the cell to obtain higher contrast of actin filaments, which is stiffer than the plasma membrane. This allowed visualization of individual actin filaments in various cell types. The detailed analyses of obtained images elucidated several kinetic parameters in a living COS-7 cell such as elongation rate and the frequency of new filament assembly. By using inhibitors and overexpression of actin- binding proteins, a dynamic equilibrium between G-actin and F-actin in the cortex has been elucidated, which is different from that of other actin-based architectures.

Combining this system with the other microscopic techniques, I tried to examine how transcription co-factor YAP, a well-known mechano-responsive regulator, controls cortical actin structures. HS-AFM analyses and other mechanical characterizations of YAP-knock out cells revealed that, the depletion of YAP increased the density of actin filaments, the stiffness of the cell cortex, and resistance against external damages. Additional biochemical and microscopic analyses suggested the involvement of E- cadherin, one of the genes regulated by YAP, in recruiting one of the Rho GTPase- activating enzymes, ARHGAP18, to the cell cortex, which inactivates RhoA. All of these results demonstrated a linkage between transcriptional control by YAP and structural changes of cortical actin via Rho GTPases, and demonstrated that HS-AFM-based visualization approach is a powerful tool to analyze the dynamics of cortical actin network and the function of regulatory proteins.

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