Efforts in this proposal focus on determining the mechanisms of regulation of vertebrate myosin-I. Myosin-I isoforms are the single-headed, membrane-associated members of the myosin superfamily that are found in most eukaryotic cells. Myosin-Is comprise the largest unconventional myosin family found in humans (eight genes). The large size and expression profile of the vertebrate myosin-I family distinguishes it as one of the most diverse classes. Myosin-Is play essential roles in membrane dynamics, cytoskeletal structure, mechanical signal-transduction, endosome processing, and possibly nuclear transcription. We are only now learning the molecular roles and modes of regulation of this important class of motors. Therefore, our efforts in this proposal focus on understanding the physical properties of vertebrate myosin-I isoforms and the regulation of these properties. We will use a combination of cell biological, biophysical, and biochemical techniques to investigate the following specific aims: 1. Biochemical and cellular interactions of myosin-I with lipid membranes. We will investigate the specificity of myosin-I isoforms for signaling lipids, we will determine the kinetics of phosphoinositide binding and release, and we will determine the motile interactions of myosin-I isoforms with membranes. 2. Microfilament-based regulation of myosin-I. Non-muscle tropomyosin plays an important role in regulating myosin-I localization and activity, but very little is known about this fundamental mode of regulation. We will investigate the biochemical details of the tropomyosin and myosin-I interaction with the goal of understanding the cellular regulation of myosin-I by microfilaments. 3. Characterization of myosin-I as a tension-sensor. Myosin-I is a low-duty ratio motor in the absence of load. However, our new investigations show that under small loads, the lifetime of actin attachment is increased dramatically, such that it becomes a high duty ratio motor. We will investigate the role of force and motor density in regulating processive motility, and we will investigate the mechanical diversity within the myosin-I family.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Deatherage, James F
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University of Pennsylvania
Schools of Medicine
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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
McIntosh, Betsy B; Ostap, E Michael (2016) Myosin-I molecular motors at a glance. J Cell Sci 129:2689-95
Pyrpassopoulos, Serapion; Arpa?, Göker; Feeser, Elizabeth A et al. (2016) Force Generation by Membrane-Associated Myosin-I. Sci Rep 6:25524
Greenberg, Michael J; Arpa?, Göker; Tüzel, Erkan et al. (2016) A Perspective on the Role of Myosins as Mechanosensors. Biophys J 110:2568-76
Kee, Anthony J; Yang, Lingyan; Lucas, Christine A et al. (2015) An actin filament population defined by the tropomyosin Tpm3.1 regulates glucose uptake. Traffic 16:691-711
Greenberg, Michael J; Lin, Tianming; Shuman, Henry et al. (2015) Mechanochemical tuning of myosin-I by the N-terminal region. Proc Natl Acad Sci U S A 112:E3337-44
Shuman, Henry; Greenberg, Michael J; Zwolak, Adam et al. (2014) A vertebrate myosin-I structure reveals unique insights into myosin mechanochemical tuning. Proc Natl Acad Sci U S A 111:2116-21
Ayloo, Swathi; Lazarus, Jacob E; Dodda, Aditya et al. (2014) Dynactin functions as both a dynamic tether and brake during dynein-driven motility. Nat Commun 5:4807
Greenberg, Michael J; Shuman, Henry; Ostap, E Michael (2014) Inherent force-dependent properties of ?-cardiac myosin contribute to the force-velocity relationship of cardiac muscle. Biophys J 107:L41-4
Zwolak, Adam; Yang, Changsong; Feeser, Elizabeth A et al. (2013) CARMIL leading edge localization depends on a non-canonical PH domain and dimerization. Nat Commun 4:2523

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