Richard J. McKenney, Ph.D. - US grants

DESCRIPTION (provided by applicant): Tuning of the Biophysical Parameters of Cytoplasmic Dynein 2 for Intraflagellar Transport Dyneins are large and complex molecular motors that transport cargo inside of living cells. There are two main classes of dyneins, the more well characterized dynein 1 family and the less understood dynein 2 family. While dynein 1 has a broad range of intracellular functions, dynein 2's role is restricted to transport within specialized cell extensions called cilia and flagella. The long-term objective of this proposal is to fully characterize the dynein 2 motor at the single molecule level and provide a genetic system for manipulating the motor in living organisms in order to determine the functional consequences of these single molecule properties. Because cilia are implicated in human diseases such as polycystic kidney disease and retinal degeneration, and dynein 2 is essential for normal functioning of these organelles, this proposal is directly relevant to human health. The proposal is divided into two core aims: the in vitro characterization of the dynein 2 motor properties at the single molecule level and the in vivo manipulation of these measured properties in the genetic model Caenorhabditis elegans (C. elegans). We will clone out a minimal dynein 2 motor domain from C. elegans and express the protein using the baculovirus system. We will characterize the enzymatic and motile properties of dynein 2 using a combination of biochemical and biophysical approaches. Parameters such as ATPase, stall force, processivity and velocity will be measured in vitro and mutations will be sought to modulate these single molecule properties. We will then reintroduce the mutated dynein 2 protein into a null background in C. elegans. This will allow us to determine how distinct biophysical properties relate to dynein 2's functions in vivo. Changes in single molecule parameters such as force production and processivity will be assayed for the first time in a living system to determine how critical those parameters are for normal motor function. PUBLIC HEALTH RELEVANCE: Ciliopathies are a broad spectrum of human diseases caused by malfunctions of cell protrusions called cilia. Cilia are built and maintained by specialized transport proteins whose functions are incompletely understood. This project will investigate, in detail, one of those transport proteins to determine how it works as a molecular motor and how its motor functions are related to the normal functioning of cilia. )

? DESCRIPTION (provided by applicant): This is a resubmission application for the K99 Pathway to Independence Award. Molecular motor proteins actively transport intracellular cargos in a highly regulated fashion. Cytoplasmic dynein is the largest, most complex, and least understood of the microtubule motor protein families. Because dynein is utilized by the cell for many diverse functions, its activity is highly regulated by a complex web of external protein factors that impinge on the basic mechanochemistry of the motor. As a graduate student, I trained with Dr. Richard Vallee at Columbia University to study the biochemistry and biophysics of cytoplasmic dynein regulation by the neurodevelopmental disease proteins LIS1 and NudE/L. During my postdoctoral career in the lab of Dr. Ronald Vale at the University of California, San Francisco, I have developed new skills in fluorescence single-molecule microscopy. I have used these new skills to discover a novel mode of dynein motility used to slide anti-parallel microtubules apart, a function critical during mitotic spindle assembly. Recently, I have utilized these skills and training to isolate and characterize a stable super-complex of dynein, dynactin, and the adapter protein BicD2 that is over 2MDa in size. I have made the first biophysical measurements of this complex at the single molecule level, revealing unanticipated new motile properties. The goal of this proposal is to understand how dynein regulatory pathways exert proper control of the motor at both the molecular and cellular level. During the mentored K99 phase, this multi-disciplinary proposal aims to: 1) Elucidate the molecular mechanism of dynein motor regulation by divergent regulatory pathways made up of dynactin-BicD2, and Lis1-NudE/L, and 2) Probe the roles of this regulatory activity in the transport of physiological important cargo in a living neuronal system. With the new training and skills acquired in the mentored phase, I will then extend the scope of my research in the independent R00 phase in an effort to understand how dynein processivity regulation is utilized in the coordination of opposite polarity motors, and what effects human neurodegenerative disease mutations have on this coordination. For the experiments proposed in this application, I will acquire additional training in nanometer-precision, multi-color single molecule microscopy and data analysis, Drosophila genetics, DNA nanotechnology, genome engineering, and confocal microscopy in live animals. As co-mentors, Ron Vale and Yuh-Nung Jan will provide expertise in advanced imaging techniques, Drosophila neurobiology and genetics. My collaborators will provide the necessary experience and support in optical trapping microscopy and novel Drosophila genome editing techniques. These new skills and training will afford me the best opportunity to achieve my career goal to launch an independent and successful research laboratory within two years. Overall, the implementation of this proposal will answer long-standing questions in the molecular transport field, and provide novel insight into the mechanism of human neurodegenerative diseases that result from impaired intracellular transport.

Title: Coordination of molecular motor activity in intracellular transport and assembly of cytoskeletal architecture. P.I. ? Richard J. McKenney Research Summary Intracellular transport is essential for cellular homeostasis in eukaryotes. Much of this process is carried out by molecular motors that convert the chemical energy from ATP hydrolysis into motion along the actin and microtubule cytoskeletal networks. Decades of research has uncovered structural and molecular details that explain how many of these motors move along their filament tracks in isolation. In the cellular milieu, most of these motors act in concert with complex regulatory machinery that links them to their respective cargos, modulates their motile properties, and dictates spatiotemporal activity. How individual motor output is controlled by this machinery is currently not clear and difficult to dissect in the complex environment of the cell. In addition, many cargos are moved simultaneously by motors of opposite polarity, in a process called bidirectional transport. How individual motors are recruited to cargo, activated, and integrated with other classes of motors presents a large challenge to the field. Further, the activities of disparate motors are harnessed to build and maintain critical cytoskeletal structures such as the mitotic spindle, cilium, and cleavage furrow. How motor and regulatory activities are coordinated to drive the self-assembly of such structures is currently a significant barrier to understanding normal and diseased cellular physiology. This application seeks to develop novel assays and tools to study the complexity of motor recruitment and regulation, bidirectional transport of cargos, and the self-assembly of cytoskeletal structures driven by motors and associated molecules. Our approach to combine biochemistry and single- molecule analysis towards in vitro reconstitutions that test molecular function, and translate our findings into in vivo systems that test hypotheses generated by these reconstitutions, will open up fruitful long-term avenues of research. We propose to: 1) Reconstitute and study the recruitment, regulation, and motility of cytoplasmic dynein and kinesin motors bound to membranous cargo through the endogenous Rab GTPase machinery that is known to link these motors to endocytic vesicles and mitochondria in cells, and 2) Reconstitute and study functions of dynein and kinesin motors that drive the self-assembly of the mitotic spindle. These broad goals build and expand on our expertise and previous work in dissecting the regulatory mechanisms of the cytoplasmic dynein motor, and aim to provide powerful new tools useful towards dissecting complex motor function. Our work will illuminate basic molecular and cell biological principles that drive cellular homeostasis and provide insight into the pathological mechanisms that arise from molecular motor malfunction.