The Direct Measurement of the Stepping Force of a Purified Vesicle using a Single Beam Optical Trap

Abstract

The transportation of vesicles is important for the development, organization, and functioning of all cellular organisms. The failure of vesicle transportation can cause many diseases and chronic illnesses associated with the malfunctioning of these systems. The molecular motors responsible for this vesicle transportation supply the energy for locomotion by exchanging chemical energy into mechanical force. Particular vesicles, zymogen granules (ZGs), are digestive enzymes secreted by the pancreas. Recently, mass spectroscopy experiments have shown that Myosin Ib, Myosin VIIb, and Myosin Vc are found on ZGs. To understand the molecular motors involved in the vesicle trafficking, we observed the in vitro motility of purified ZGs from rat pancreas and examine the stepping manner and force that is produced using a single beam optical trap. The average speed of the ZGs is around 100 nm/sec, which is similar to our previous study of two myosin Vc molecules tagged with DNA. The stepping seems to have patterns; one is unidirectional steps with 16-nm to 72-nm step-size, while some steps have a back-and-forth ensemble stepping along the axis of actin filaments. The average force and maximum force in the optical trap are 0.3 ± 0.1 pN and 0.5 ± 0.2 pN, respectively. The typical force of a single molecular motor has a range of 1 pN to 5pN. Thus, our finding in the unidirectional movement can be created by a single motor. Our results conclud that most steps may be created by myosin Vc.