Altman:Research
Department of Physics, Willamette University
Studying the regulated activity of myosin motors
Motor proteins are molecules in the cell that convert chemical energy into mechanical energy, and are thus important in numerous cellular processes that require generation of motion and force. We investigate myosin, a motor protein superfamily involved in processes such as muscle contraction, cell division, intracellular vesicle transport, and cell migration. We use in vitro and in vivo assays to study the regulated function of a variety of myosins
Single molecule studies of myosins
We are examining the force-sensitivity of Acanthamoeba myosin 1c (AM1C) activity. Class 1 myosins have been split into two subclasses. While subclass 2 myosins are hypothesized to have a force-dependent activity, subclass 1 myosins are not. Two subclass 2 myosins (Rat Myo1b and Mouse Myo1c) have been shown to have a high degree of force-sensitivity, but no subclass 1 myosin has yet been tested. To test the force sensitivity of AM1C, which is from subclass 1, we are using an optical trap, which allows us to apply picoNewton forces to single myosin motors.
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Cartoon of a myosin attached to a bead that is held in an optical trap.
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Image taken from De La Cruz, et al.
We are conducting similar studies of myosin 6 with a mutation associated with hypertrophic cardiomyopathy (HCM) (Myosin 6 H246R). We seek to determine how the H246R mutation alters the chemomechanical cycle of the motor.
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Crystal structure of the myosin 6 motor domain and insert-2 in the pre-powerstroke state. Insert-1 and insert-2 are shown in yellow and pink, respectively, and L310 and H246 are shown as spheres that are blue and green, respectively. In addition, bound MG-ADP-AlF4 is shown as a stick model.
Retinal pigment epithelium phagocytosis
Retinal pigment epithelium (RPE) cells phagocytose waste shed by rod photoreceptor cells. This is important for the health of the eye, and failure to complete this process results in retinal degeneration. We study the role of molecular motors in this process. Specifically, we are testing the hypothesis that motor proteins myosin VI and VIIa generate the forces and motion required for the internalization of rod cell debris. To do this, we observe internalization of micron-sized microspheres by a primary RPE cell line (ARPE-19). To perturb the function of a particular myosin, we over-express myosins lacking the motor domain.
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Overlay of a DIC and fluorescence image of an ARPE-19 cell internalizing microspheres while transiently over-expressing a GFP-tagged myosin
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Tracking a 1-micron-diameter microsphere that has been internalized by an ARPE-19 cell.
Techniques used in the lab
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Example of motility of fluorescent actin filaments driven by the myosin VI motors on the experimental surface. Total duration of movie is 10 minutes.
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7.5 minute movie of a fluorescent bead in an ARPE-19 cell that is being trafficked toward the nucleus. Scale bar is 1 um.
