Class V myosins (myoV) play crucial roles in actin-based organelle transport and membrane trafficking. There are three mammalian isoforms (Va, Vb, and Vc) with different tissue specificities and functions. Two of these isoforms (Va and Vb) are processive, meaning that they can take multiple steps on actin before dissociating. MyoVc is non-processive, yet all function in cargo transport. Much attention has been given to the in vitro study of single motor motion on individual actin filaments, primarily using a constitutively active truncated version of myoVa. An overarching goal of this proposal is to systematically build complexity into the study of class V myosin motors, using three complementary approaches. The first is to investigate the properties of the full-length motor, and how cargo impacts on its function. The second is to gain insight into how myoV motors work together, because cellular cargo is transported by multiple motors. The third is to introduce myoV motors into cells and track their motion, or the motion of native cargo driven by motors with known properties. In this context, we will assess differences between the three myoV isoforms.
In Aim 1 we will determine how the single molecule properties of regulated full- length myoVa differ from those of constitutively active constructs, in the absence or presence of cargo. Run lengths, speed, and stepping patterns of single full-length myoVa motors, in the absence or presence of cargo, will be determined. We will test if cargo adapter proteins affect motor function beyond initial activation.
In Aim 2 we investigate how two myoV motors co-ordinate their motion and force generation under unloaded and loaded conditions. DNA scaffolds that bind exactly two motors will be used to determine the effect that myoV motors have on each others'properties, under unloaded and loaded conditions. Parameters to be measured are speed, run length, step size, and stall force. Various combinations of motors will be tested. The results have implications for the behavior of multiple motors that are mechanically coupled by being bound to the same intracellular cargo.
In Aim 3 we compare and contrast the motion of myoVa, myoVb, and myoVc within a living cell. The trajectories of Quantum-dot (Qdot) labeled myoV constructs, introduced into mammalian cells by pinocytosis, will be followed by high resolution total internal reflection fluorescence (TIRF) microscopy and single particle tracking. Movement of the two processive myoV isoforms (myoVa and myoVb) will be compared to non-processive myoVc. Different cell types with varying actin architectures will be used. A complementary inducible cargo trafficking assay will be used to target motor constructs to native cargo. We believe that a combined approach as presented here provides the greatest potential to move the field forward.

Public Health Relevance

Class V myosins (myoV) play crucial roles in actin-based organelle transport and membrane trafficking, and are needed for survival in both mammals and lower organisms. Mutations in myoVa lead to Griscelli syndrome, while mutations in myoVb cause vascular inclusion disease. Understanding how these motors work at a molecular level will contribute to informed design of new preventative or therapeutic interventions for these diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gindhart, Joseph G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Vermont & St Agric College
Schools of Medicine
United States
Zip Code
Barua, Bipasha; Sckolnick, Maria; White, Howard D et al. (2018) Distinct sites in tropomyosin specify shared and isoform-specific regulation of myosins II and V. Cytoskeleton (Hoboken) 75:150-163
Sladewski, Thomas E; Billington, Neil; Ali, M Yusuf et al. (2018) Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex. Elife 7:
Rynkiewicz, Michael J; Prum, Thavanareth; Hollenberg, Stephen et al. (2017) Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function. Biophys J 113:2444-2451
Krementsova, Elena B; Furuta, Ken'ya; Oiwa, Kazuhiro et al. (2017) Small teams of myosin Vc motors coordinate their stepping for efficient cargo transport on actin bundles. J Biol Chem 292:10998-11008
Zimmermann, Dennis; Homa, Kaitlin E; Hocky, Glen M et al. (2017) Mechanoregulated inhibition of formin facilitates contractile actomyosin ring assembly. Nat Commun 8:703
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
Pollard, Luther W; Bookwalter, Carol S; Tang, Qing et al. (2017) Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation. Proc Natl Acad Sci U S A 114:E7236-E7244
Tang, Qing; Billington, Neil; Krementsova, Elena B et al. (2016) A single-headed fission yeast myosin V transports actin in a tropomyosin-dependent manner. J Cell Biol 214:167-79
Sckolnick, Maria; Krementsova, Elena B; Warshaw, David M et al. (2016) Tropomyosin isoforms bias actin track selection by vertebrate myosin Va. Mol Biol Cell 27:2889-97
Sladewski, Thomas E; Krementsova, Elena B; Trybus, Kathleen M (2016) Myosin Vc Is Specialized for Transport on a Secretory Superhighway. Curr Biol 26:2202-7

Showing the most recent 10 out of 37 publications