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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Gindhart, Joseph G
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University of Vermont & St Agric College
Schools of Medicine
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Powell, Cameron J; Ramaswamy, Raghavendran; Kelsen, Anne et al. (2018) Structural and mechanistic insights into the function of the unconventional class XIV myosin MyoA from Toxoplasma gondii. Proc Natl Acad Sci U S A 115:E10548-E10555
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Whitelaw, Jamie A; Latorre-Barragan, Fernanda; Gras, Simon et al. (2017) Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion. BMC Biol 15:1
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Lombardo, Andrew T; Nelson, Shane R; Ali, M Yusuf et al. (2017) Myosin Va molecular motors manoeuvre liposome cargo through suspended actin filament intersections in vitro. Nat Commun 8:15692
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Ali, M Yusuf; Vilfan, Andrej; Trybus, Kathleen M et al. (2016) Cargo Transport by Two Coupled Myosin Va Motors on Actin Filaments and Bundles. Biophys J 111:2228-2240
Michalek, Arthur J; Kennedy, Guy G; Warshaw, David M et al. (2015) Flexural Stiffness of Myosin Va Subdomains as Measured from Tethered Particle Motion. J Biophys 2015:465693
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

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