Setting up an In Vitro Reconstitution System to Study Actomyosin-Driven Membrane Organisation

Abstract

The plasma membrane is the site of numerous crucial processes like signal transduction, cell adhesion and vesicular transport. These processes require the plasma membrane to be organised into distinct meso and nanoscale domains. Prevailing models of membrane organisation have neglected the role of the membrane-associated actin cortex in generating such domains through active, non-equilibrium processes. These models instead focus on passive, equilibrium processes emerging from protein-protein, lipid-protein and lipid-lipid interactions. The Active Composite model emerged as an explanation for the non-equilibrium properties shown by certain membrane components like GPI-anchored proteins, which are largely inconsistent with the current paradigm of membrane organisation. The model predicts that actin-bound membrane components can undergo transient, active clustering as a consequence of ATP-driven actomyosin contractility. Previous work has shown that such clustering can occur in vitro due to the activity of skeletal muscle myosin II on pre-polymerised actin filaments. However, some caveats exist with this work. Actin filaments in cells are dynamic and undergo continuous treadmilling rather than being of fixed length. Moreover, skeletal muscle myosin II is a specialised motor that operates primarily in muscle cells. Non-muscle myosin II, on the other hand, is responsible for cytoplasmic actin contractility across eukaryotic cell types. The two myosins also differ in their length scales and motor activity kinetics. Broadly, the aim of this thesis is to build an in vitro reconstitution system that better captures the dynamic nature of the membrane-associated actomyosin cortex by incorporating spontaneously polymerising dynamic actin filaments along with non-muscle myosin II motors. Such a system would be more suited to test the predictions of the Active Composite model. In this direction, I attempted to recombinantly express non-muscle myosin II as well as reconstitute spontaneously polymerising actin filaments on a supported lipid bilayer.