Measuring the Force of Single and/or Multiple Myosin 5 by Using a Single Beam Optical Trap

Abstract

The transportation of vesicles is important for the development, organization, and functioning of all cellular organisms. The failures of vesicle transportation cause many diseases and chronic illnesses associated with the malfunctioning of these systems. The molecular motors responsible for these vesicle transportations supply the energy for locomotion by exchanging chemical energy into mechanical force. To measure the forces generated by these molecular motors we use an optical trap to capture polystyrene beads that have myosin molecules attached to their surface. To control the number of myosin on the surface, the concentration of myosin can be changed. This will be a stepping stone to studying a particular transport vesicle, zymogen granules, which are large digestive enzymes secreted by the pancreas. Some fundamental questions exist as to how these vesicles move throughout the cell. Some motor proteins are processive (myosin 5A), meaning that they take several steps before falling off. Others are non-processive (myosin 5C). Our lab has shown that two myosin 5c molecules attached to a DNA scaffold can move processively, suggesting that perhaps multiple myosin 5c may work together to transport extremely large vesicles. Investigating the mechanism of transportation and the force generation involved can be done by combining the use of fluorescence imaging and optical tweezer techniques. In order to model the forces involved in vesicle transport, modified polystyrene beads (diameter: 200 nm ∼ 3 µm) with motor proteins such as myosin 5 can be held in an optical trap to calculate the forces generated. These force measurements can be used to estimate the number of motors involved for processive movement along actin filaments.