Myosin Va (myoVa) and myosin VI (myoVI) are double-headed, processive molecular motors that transport intracellular cargo over long distances by way of actin filament tracks. With their ability to travel in opposite directions along the polarized actin cytoskeleton, myoVa transport is critical for exocytosis while myoVI is associated with endocytosis. To successfully deliver cargo, both myoVa and myoVI must overcome physical challenges presented by the intracellular milieu and the interactions with other motors that are attached to the same cargo. To assess these motors'inherent transport capabilities, we will use state-of-the- art single molecule biophysical techniques with high spatial (>6nm) and temporal (<2ms) resolution to characterize the stepping dynamics of single myoVa or myoVI motors, independently or when linked together in a tug of war.
Aim #1 will determine how the individual heads of a myoVa molecule coordinate and adjust their stepping behavior to maintain processive motion in response to both resistive and assistive forces. This will be accomplished by monitoring the individual heads that are labeled with different color quantum dots (Qdots) as force is applied to the motor in a combination laser trap-TIRF microscope.
In Aim #2, myoVI will be similarly characterized. However, myoVI is unique in that it takes unexpectedly large steps, challenging the "swinging lever arm" model. Through structural mutagenesis, we will determine whether the proximal and/or medial tail serve as surrogate lever arms to allow myoVI to step processively. Finally in Aim #3, we will build complexity in vitro by linking myoVa and myoVI motors to the same Qdot cargo, as a model for intracellular cargo transport by multiple motors. We will unambiguously determine the stepping dynamics of both motors simultaneously as they attempt to transport the same cargo. With the data gathered in Aims #1 and #2, we will quantitatively model and predict the outcome of these tug of war scenarios as it relates to intracellular bidirectional transport by ensembles of molecular motors.

Public Health Relevance

The transport of intracellular cargo is one of the most basic cellular processes that relies on tiny molecular motors such as myosin Va and myosin VI to deliver cargoes ranging from insulin granules to endocytic vesicles, respectively. With genetic mutations in the myo5a gene resulting in neurological impairment or immunodeficiency and myo6 gene mutations leading to hypertrophic cardiomyopathy, understanding the normal function of these motors has major implications for therapeutic management of these genetic disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM094229-04
Application #
8641390
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gindhart, Joseph G
Project Start
2011-04-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
4
Fiscal Year
2014
Total Cost
$294,310
Indirect Cost
$67,830
Name
University of Vermont & St Agric College
Department
Physiology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Nelson, Shane R; Trybus, Kathleen M; Warshaw, David M (2014) Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va. Proc Natl Acad Sci U S A 111:E3986-95
Mukherjea, Monalisa; Ali, M Yusuf; Kikuti, Carlos et al. (2014) Myosin VI must dimerize and deploy its unusual lever arm in order to perform its cellular roles. Cell Rep 8:1522-32
Heaslip, Aoife T; Nelson, Shane R; Lombardo, Andrew T et al. (2014) Cytoskeletal dependence of insulin granule movement dynamics in INS-1 beta-cells in response to glucose. PLoS One 9:e109082
Ali, M Yusuf; Previs, Samantha B; Trybus, Kathleen M et al. (2013) Myosin VI has a one track mind versus myosin Va when moving on actin bundles or at an intersection. Traffic 14:70-81
Sckolnick, Maria; Krementsova, Elena B; Warshaw, David M et al. (2013) More than just a cargo adapter, melanophilin prolongs and slows processive runs of myosin Va. J Biol Chem 288:29313-22
Armstrong, Jessica M; Krementsova, Elena; Michalek, Arthur J et al. (2012) Full-length myosin Va exhibits altered gating during processive movement on actin. Proc Natl Acad Sci U S A 109:E218-24
Warshaw, David M (2012) Tilting and twirling as myosin V steps along actin filaments as detected by fluorescence polarization. J Gen Physiol 139:97-100
Nelson, Shane R; Ali, M Yusuf; Warshaw, David M (2011) Quantum dot labeling strategies to characterize single-molecular motors. Methods Mol Biol 778:111-21
Ali, M Yusuf; Kennedy, Guy G; Safer, Daniel et al. (2011) Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport. Proc Natl Acad Sci U S A 108:E535-41