VESICLE TRANSPORTATION OF ZYMOGEN GRANULE BY MYOSIN; FORCE MEASURING THE STEPPING BY OPTICAL TWEEZER

           Zymogen granule is an enzymatic vesicle in the pancreas. The surface of the zymogen granules (ZGs) has several different myosin classes, such as myosin Ib, VI, VIIb, and Vc. These molecular motors may contribute ZG’s transportation in alpha-cells. To understand what the molecular motors involved in the vesicle trafficking, we observed the in vitro motility of the purified ZGs from rat pancreas and exam the stepping manner and force that is used by a single beam optical tweezer. The average force and maximum force from myosins on the ZGs are 0.3 ± 0.1 pN and 0.5 ± 0.2 pN, respectively. A typical force of a single molecular motor was a range of 1 pN to 5pN. The average step-size is 35 nm, which coincides with the actin-helical repeat. A stepping traces were detected within the range of force. We expect that each step was exerted by a single motor. However, different myosins may be able to generate force simultaneously. To understand which molecular motor exerted a force on ZGs, myosin inhibitor, PCIP and TIP, were used in this study. Less than 10 mM of and more than 100 mM of PCIP inhibits motor activities of myosin Ic and Vc, respectively, but does not inhibit myosin VI and VIIb. TIP (10~100 mM) inhibits Myosin VI activity, but does not inhibit other myosin's activity. Using only PCIP or TIP or a combination of PCIP/TIP, stepping movement of ZGs by using the optical trap has been observed. Successful results will present about the directionality of the movement as well as stepping kinetics.