Counting Molecules in Non-Muscle Myosin II Filaments

Abstract

Non-muscle (NM) myosin II is involved in many important cellular processes, including migration, adhesion and cytokinesis. NM myosin II assembles into bipolar filaments; this multimerization is thought to be essential for acto-myosin contractility. Since each head in a myosin filament can bind F-actin, the number of heads per filament will affect the balance among its bundling, crosslinking and sliding activities, as well as the load-dependent feedback on these activities. Electron microscopy studies of interphase cells from different species have shown NM myosin II filaments contain 16-56 heads, but the size of filaments has never been determined in cytokinesis.We first studied the number of NM-myosin II in meiotic and mitotic C. elegans zygotes. GFP tagged myosin was imaged by TIRF microscopy until the fluorescence signal was completely bleached. We developed a novel, yet very simple molecule counting method CoMPaS (counting molecules with photobleaching and subtraction) to measure the number of bleaching events in the fluorescence decay curves from cells with different level of GFP expression. We found that the total number of myosin molecules in a single filament is 100 - 150. As an independent assessment of myosin filament size, we also measured filament length in cultured Drosophila cells using super-resolution microscopy. We measured the distance between diffraction-limited foci containing GFP-tagged regulatory light chain (RLC) at the two ends of myosin filaments, with nm accuracy. On average, myosin filaments are approximately 380 nm long. This method also allowed us to determine the orientation and size of the RLC myosin head foci. Interestingly, increased myosin filament length correlated with more parallel filament head foci. We will combine these measurement techniques with genetic perturbations to understand how NM myosin II filament assembly is controlled and how interaction with actin influences myosin filament structure.