Transport processes within the cell rely on carrier proteins such as the molecular motor myosin VI, a myosin motor protein with the unique ability to carry cargo towards the minus end of actin filaments. Although myosin VI has been implicated in both the secretory and endocytic trafficking pathways, live cell investigation of the specifics of its roles in these pathways has been lacking. As such, we have used a unique, live-cell secretion assay to investigate the specific roles of myosin VI and its binding partner optineurin in the secretory pathway. During constitutive secretion, proteins synthesized at the endoplasmic reticulum (ER) are transported to the Golgi complex for processing and then to the plasma membrane for incorporation or extracellular release. Small interfering RNA-based knockdown of myosin VI causes an ER-to-Golgi transport delay, suggesting an unexpected function for myosin VI in the early secretory pathway. Depletion of myosin VI or optineurin does not affect the number of vesicles leaving the trans-Golgi network, indicating that these proteins do not function in trans-Golgi vesicle formation. However, myosin VI and optineurin colocalize with secretory vesicles at the plasma membrane. Furthermore, live-cell total internal reflection fluorescence (TIRF) microscopy demonstrates that myosin VI or optineurin depletion reduces the total number of vesicle fusion events at the plasma membrane and increases both the proportion of incomplete fusion events and the number of docked vesicles in this region. These results suggest a novel role for myosin VI and optineurin in regulating the fusion pores that are formed between secretory vesicles and the plasma membrane during the final stages of secretion. To complement these studies on the role of myosin VI in secretion, we used live cell fluorescence recovery after photobleaching (FRAP) to compare the turnover rates of myosin VI on the clathrin-coated vesicles and early endosomes of the endocytic uptake pathway. These data offer novel insight into the kinetics of myosin VI in the endocytic pathway and the general nature of the turnover of a motor protein and its binding partners on specific intracellular structures, by demonstrating differences in turnover between wildtype myosin VI and an artificially dimerized myosin VI construct, a deafness mutant of myosin VI (D179Y), and the myosin VI binding partner Dab2. Overall, this examination of myosin VI in the secretory and endocytic pathways enhances our understanding of the basic, biomechanical operations of the cell and offers unique insights into the etiology of diseases stemming from mutations in myosin VI, such as hypertrophic cardiomyopathy and neurodegeneration.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
National Heart, Lung, and Blood Institute
Zip Code
Heissler, Sarah M; Selvadurai, Jayashankar; Bond, Lisa M et al. (2012) Kinetic properties and small-molecule inhibition of human myosin-6. FEBS Lett 586:3208-14
Bond, Lisa M; Brandstaetter, Hemma; Sellers, James R et al. (2011) Myosin motor proteins are involved in the final stages of the secretory pathways. Biochem Soc Trans 39:1115-9
Bond, Lisa M; Peden, Andrew A; Kendrick-Jones, John et al. (2011) Myosin VI and its binding partner optineurin are involved in secretory vesicle fusion at the plasma membrane. Mol Biol Cell 22:54-65