Myosin-6 Mobility At The Plasma Membrane Of Cultured Mammalian Cells

Myosin-6 is a reverse-directed, actin-based, molecular motor responsible for moving cellular cargo towards the minus-end of actin filaments. It is involved in clathrin-mediated endocytosis and intracellular transport. We have used TIRF microscopy to study the movement and localisation of the “short insert” isoform of eGFP-myosin-6 within live 3T3 fibroblasts and human endothelial cells. Individual GFP tagged myosin-6 particles were automatically tracked by computer. Diffusion of membrane associated proteins is usually controlled by lipid mobility and individual trajectories follow a simple “random walk” in which mean squared displacement (MSD) is proportional to time interval (dT) and the lateral diffusion coefficient. We found the movement of myosin-6 shows anomalous diffusive behaviour and Dlat values are better described by a single exponential function, with a significant fraction of molecules (∼30%) having Dlat < 0.02 μm2 s−1. Close examination of individual trajectories revealed that myosin-6 molecules exhibit periods of free movement interspersed with intervals in which motion is arrested. This phenomenon has been described previously as “transient confinement”. The length of time during which movement of myosin-6 molecules were arrested had an exponential lifetime distribution (t1/2 ≈ 250 ms). The number of observed particles decreased exponentially with time due to photobleaching. However, the shape of the intensity distribution of the individual particles remained constant and was well described by a fit to the sum of two Gaussian terms. We conclude that myosin-6 molecules associate with the plasma membrane and bind intermittently with immobile sub-cellular structures, which might be either the actin cytoskeleton or trans-membrane anchoring proteins. The “short insert” splice variant of myosin VI is predominantly monomeric.