Access Type

Open Access Dissertation

Date of Award

January 2022

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physics and Astronomy

First Advisor

Takeshi Sakamoto

Abstract

Zymogen granules are enzymatic vesicles in the pancreas. The surface of these zymogen granules (ZGs) has several different kinds of myosin molecules, such as myosin 1c, 6, 5c, and 7b. These molecular motors may contribute to ZG transportation in cells. To understand the molecular motors involved in the vesicle trafficking, we observed the in vitro motility of purified ZGs from rat pancreas and examined the stepping behavior and force that is generated using a single beam optical trap. To be involved in trafficking, molecular motors have certain characteristics, a high duty ratio and the ability to move continuously along actin tracks. The high duty ratio means that during most of the ATPase cycle the motor is in a bound state with actin. Interestingly, Myosin 1c and 5c are low duty ratio motors, meaning that the molecule itself cannot move continuously along the actin filament. Recently our laboratory demonstrated that two myosin 5c molecules, when tethered with DNA origami, move continuously and suggested that more than two molecules of myosin 5c may be able to transport the ZGs. Besides, the surface curvature of ZGs (diameter: 1 m) is much flatter than that of a neuronal vesicle (diameter: 40 nm) that is transported by a high duty ratio motor, myosin 5a. In this case, although myosin 1c and 5c are low duty ratio motors, these motors may be able to transport the ZGs. We found that the complicated stepping of ZGs allow for movement using an ensemble of motors, even non-processive motors, that when acting together on the ZG surface display movement and kinetics much different than single molecule studies. Additionally, we attempt to explore the impact of myosin 6, a backwards stepping motor, on the movement in this ensemble of motors.

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