David M. Warshaw, Ph.D.
Brandon Bensel, Ph.D.
Kinesin and myosin molecular motors
Proper delivery of vesicular cargoes via intracellular transport is necessary to ensure healthy cellular function. To date, much work has been done to understand the biophysics of molecular motor-based cargo transport in vitro, yet much of this work has been done in 2 dimensions with a single type of motor on a rigid cargo. However, in vivo vesicular transport occurs in 3-dimensional cytoskeletal networks, and vesicular cargoes are often decorated with multiple copies of multiple types of molecular motor. My work aims to understand how teams of multiple kinesin and myosin molecular motors navigate complex 3-dimensional networks of microtubules and actin filaments while bound to fluid-like liposome cargoes. I use purified recombinant motor proteins, purified tubulin and actin, and lab-made liposomes to reconstitute a complex in vitro model system of vesicular cargo transport on a microscope slide. 3D STORM and single-particle tracking are used to characterize cytoskeletal networks and monitor liposome motion with nanometer spatial resolution and <100-millisecond temporal resolution. I also use optical trapping to assess force generation by motor ensembles, and TIRF microscopy for 2D motility experiments. Together these techniques allow me to understand the emergent properties of cargo transport in a more physiological system as compared to classical single molecule studies.
Shane R. Nelson, Ph.D.
Andrew Mead, Ph.D.
Muscle powers a diverse range of biological functions in vertebrates, from the rapid vocalizations of songbirds to the slow swimming of carp. Remarkably, muscle’s basic force-generating structure, the sarcomere, is highly conserved among vertebrate muscles, as are the genes that encode its constituent proteins. I use the swimming muscles of zebrafish larvae as a model system to ask basic questions about how sarcomeres produce force and motion, and in particular how their mechanical characteristics can be ‘tuned’. Larval muscles develop and begin to function within a few days of fertilization, meaning that I can generate experimental muscles, with genetic modifications or other interventions, comparatively easily and rapidly. The orientation of muscle fibers within the larval tail allows me to apply suite of classical muscle mechanics experiments to quantify the effects of these interventions on muscle force, shortening velocity, and power. My current work focuses on Myosin Binding Protein C (MyBP-C), which is understood to be an important regulator of cardiac and skeletal muscle function. Single molecule biophysical studies by the Warshaw group and others have begun to uncover the molecular mechanisms by which the various isoforms of MyBP-C may affect muscle mechanics. By expressing these isoforms in zebrafish we can observe how these molecular insights play out in the higher order setting of an intact, living muscle.
Samantha Previs
I am involved in experiments looking at the function of myosin-binding protein C and its role in cardiac muscle contraction, insulin granule secretion, and myosin transport on actin networks. I also provide general laboratory support by preparing reagents and ordering needed supplies.
Sebastian Duno-Miranda
Angela Ploysangngam
Guy Kennedy
My goal is to design, develop, fabricate and support world class microscopy systems for research in molecular physiology. Our research demands high sensitivity with fast temporal and precise spatial resolution. These systems include integrated Laser Optical Tweezers with dual color Total Internal Reflection Microscopy (TIRFM) along with single molecule detection, manipulation and tracking capabilities. Mechanical measurements of pico-newton force, nanometer displacement, and pico-newtons/ nanometer stiffness are used to address questions in single molecules. These techniques are applied to protein ensembles, intercellular dynamics, and molecular protein- protein interactions. High speed fluorescent imaging of single molecule dynamics are possible using state of the art ICCD cameras with TIR and far field illumination. Recent initiatives include high speed 3D tracking, STORM, and PALM Super Resolution microscopy. Myosin, Kinesin, C-Protein, Actin, Microtubules and other Cytoskeletal proteins are studied with our techniques.