Active transport of vesicular cargoes is vital to the targeted delivery of organelles, proteins, and signaling mol- ecules in the complex and crowded cellular environment. Accordingly, defects are linked to developmental, neurodegenerative, pigmentation, immunological, and other diseases. Knowing the detailed mechano- chemistry and structural dynamics of isolated motor proteins is essential for integrating the divergent mechani- cal and kinetic properties of cytoskeletal motor families. We have developed a number of powerful new bio- physical tools that reveal the modulation of mechanical and ATPase reaction kinetics under applied mechani- cal force, elucidate the essential rotational conformational transitions of specific domains within the motor pro- teins and produce reliable force, stepping dynamics, and local viscoelastic parameters of the cellular environ- ment. We will apply these unique tools to investigate the divergent biochemical and mechanical properties of myosin-I (Myo1) and dynein isoforms that have that have not yet been approached at the mechanistic detail now possible.
Aim 1 : Using simultaneous optical trapping and TIRF microscopy, determine stress- and strain-dependence of the binding and dissociation of ATP and ADP that fuel motion and control motor stepping;
Aim 1 A: in myosins 1b and 1c;
Aim 1 B: in budding yeast and mammalian cytoplasmic dynein.
Aim 2 : To determine the rotational motions of motor heads and lever arms that generate force and cargo translocation using single molecule polarized TIRF (polTIRF) microscopy;2A: in myosins 1b and 1c;2B: in yeast cytoplasmic dynein. We discovered that Myo1b and Myo1c have markedly different strain-dependent attachment lifetime, even though their unloaded kinetics and protein sequences are quite similar. We will apply our formidable single-molecule mechanical and fluorescence technologies to understand how these closely related motors have optimized their kinetic and structural variations for their different cellular roles. Mammalian and Saccharomyces cerevisiae cytoplasmic dynein have overlapping roles in their respec- tive cells, but their properties are very different. We are in the unique position to compare them using advanced biophysical methods. We will determine their strain-dependent ATPase dynamics and rotational motions. We anticipate that these studies will provide insight into the motors'mechanisms, and will also reveal biochemical and mechanical adaptations to their distinct functions.
Aim 3 : We will use our unique new technology for measuring and calibrating optical trap signals within live cells to 3A: determine the functional speciali- zations that vary the complement of motors among early and late endosomes, and small and large la- tex bead compartments (LBCs) endocytosed or phagocytosed into live cells. 3B: measure forces and motor dynamics during remodeling of endoplasmic reticulum (ER) by interaction with endosomal vesi- cles. All three aims represent close, essential collaborations with Drs. Ostap, Holzbaur and Shuman. Many cargos have opposing motors bound simultaneously;these motors may operate in teams functioning either cooperatively or competitively. We will focus on the role of oppositely directed motors at critical junctures in cellular organelle trafficking: early and late endosomes and the remodeling of endoplasmic reticulum we have observed when transport vesicles interact with the ER. Together, these studies will provide insights into the roles of the molecular motor families in regulating organelle motility, morphology and remodeling. These stud- ies will lead to a much improved understanding of myosin I and dynein isoforms that operate very differently from the better understood kinesin and myosin motors, leading to a more rigorous understanding of their func- tions in cell biology and disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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University of Pennsylvania
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McIntosh, Betsy B; Pyrpassopoulos, Serapion; Holzbaur, Erika L F et al. (2018) Opposing Kinesin and Myosin-I Motors Drive Membrane Deformation and Tubulation along Engineered Cytoskeletal Networks. Curr Biol 28:236-248.e5
Moore, Andrew S; Holzbaur, Erika L F (2018) Mitochondrial-cytoskeletal interactions: dynamic associations that facilitate network function and remodeling. Curr Opin Physiol 3:94-100
Woody, Michael S; Capitanio, Marco; Ostap, E Michael et al. (2018) Electro-optic deflectors deliver advantages over acousto-optical deflectors in a high resolution, ultra-fast force-clamp optical trap. Opt Express 26:11181-11193
Lee, In-Gyun; Olenick, Mara A; Boczkowska, Malgorzata et al. (2018) A conserved interaction of the dynein light intermediate chain with dynein-dynactin effectors necessary for processivity. Nat Commun 9:986
Lippert, Lisa G; Dadosh, Tali; Hadden, Jodi A et al. (2017) Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk. Proc Natl Acad Sci U S A 114:E4564-E4573
Pyrpassopoulos, Serapion; Shuman, Henry; Ostap, E Michael (2017) Adhesion force and attachment lifetime of the KIF16B-PX domain interaction with lipid membranes. Mol Biol Cell 28:3315-3322
Lewis, John H; Jamiolkowski, Ryan M; Woody, Michael S et al. (2017) Deconvolution of Camera Instrument Response Functions. Biophys J 112:1214-1220
Greenberg, Michael J; Shuman, Henry; Ostap, E Michael (2017) Measuring the Kinetic and Mechanical Properties of Non-processive Myosins Using Optical Tweezers. Methods Mol Biol 1486:483-509
Hendricks, Adam G; Goldman, Yale E (2017) Measuring Molecular Forces Using Calibrated Optical Tweezers in Living Cells. Methods Mol Biol 1486:537-552
Kast, David J; Dominguez, Roberto (2017) The Cytoskeleton-Autophagy Connection. Curr Biol 27:R318-R326

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