THE ROLE OF MYOSIN VA AND THE DYNEIN/DYNACTIN COMPLEX IN NEUROFILAMENT AXONAL TRANSPORT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By: Nael H Alami Graduate Program in Molecular, Cellular, and Developmental Biology The Ohio State University 2009 Dissertation Committee: Anthony Brown, Advisor Harold Fisk James Jontes Dale Vandre Copyright by Nael H Alami ABSTRACT Neurofilaments are the major cytoskeletal elements in mature neuronal cells. They are known for their space-filling properties and for forming an elastic network along the axons that is responsible for radial growth and maintaining proper caliber. Neurofilaments and other cytoskeletal polymers, membranous organelles, and macromolecular cargo are transported along the axon in one of two directions: away from the cell body, towards the axon tips, in an anterograde fashion, or back towards the cell body from the direction of the growth cones, in a retrograde fashion. The regulation of this transport is vital for the functional and structural well-being of the neuron and is mainly dependent on the microtubule- based motor proteins kinesins and dynein/dynactin. In 2002, a study by Rao et al. suggested a role for the actin-based motor protein myosin Va in neurofilament transport. They reported that myosin Va associates with neurofilaments in vivo and that neurofilaments accumulate in axons of neurons lacking myosin Va. Based on these observations, we hypothesize that myosin Va is involved in neurofilament transport and that in the absence of myosin Va, neurofilaments move less efficiently along the axons. To test this hypothesis, we used fluorescent live-cell imaging of neurofilament movement in ii SCG neurons from wild type and dilute lethal mice. Our results indicate that the absence of myosin Va from SCG neurons does not significantly alter neurofilament velocity or frequency of movement. We also used a fluorescence photoactivation pulse-escape technique to measure the rate of departure of photoactivatable GFP-tagged neurofilaments from photoactivated axonal regions in cultured DRG neurons from two strains of dilute lethal mice. We observed a 48%-169% increase in the mean time for neurofilaments to depart the activated regions in neurons from dilute lethal as compared to wild type. We conclude that neurofilaments pause for more prolonged periods in the absence of myosin Va. We propose that myosin Va is a short-range motor for neurofilaments and that it can function to enhance the efficiency of neurofilament transport in axons by delivering neurofilaments to their microtubule tracks. We also studied the role of dynein/dynactin in neurofilament transport. Dynein/dynactin is a retrograde motor complex that was found to associate with neurofilaments in vivo and in vitro. It has been previously proposed that it is responsible for retrograde neurofilament transport but without any direct evidence. We used SCG and cortical neuronal cultures to observe neurofilament transport in cells where dynein/dynactin activity has been disrupted using a number of different approaches that target different subunits of the complex. Using dynein heavy chain knock-down, dynein intermediate chain functional blocking antibody, dynamitin/p50 overexpression in SCG neurons and p150- coiled-coil1 overexpression in cortical neurons, we report an inhibition of iii retrograde transport. This clearly indicates that dynein/dynactin is indeed the retrograde neurofilament motor. We also observed a reciprocal inhibition of anterograde transport that mirrored the retrograde transport inhibition in every one of these manipulations. This suggests that a tight functional coupling exists between the retrograde and anterograde motors of neurofilaments, where the activity of one motor is needed for the activity of the other and vice versa. In one of our observations after disrupting dynein/dynactin activity using p150-coiled-coil overexpression in SCG neurons, we report an increase in anterograde transport. Our attempts to reproduce this result in cortical neurons, at different times after transfection, or with different transfection concentrations failed, as we observe an inhibition in both directions of transport in all such cases. This unique result, therefore, remains to be explored. In conclusion, we propose that the transport and organization of neurofilaments may be orchestrated by the coordinated activity of at least three different motor proteins, kinesins, dynein/dynactin, and myosin-Va, which act together to convey and distribute these polymers along neuronal axons, and the disruption of any of these motors could lead to neurofilament transport defects and accumulations that could ultimately result in neuronal degeneration. iv To my family v ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor Dr. Anthony Brown for his invaluable advice and support. The fruits of my work are a result of his supervision and guidance, and for that I am grateful. I would also like to thank my committee members, professors Harold Fisk, James Jontes and Dale Vandre for their patience, insightful suggestions and critical review of my dissertation. Past and present members of the Brown lab have helped make the work environment throughout those past years enjoyable and exciting. I would like to thank Dr. Niraj Trivedi for his friendship and continuous support, Dr. Atsuko Uchida, Lina Wang, Paula Monsma and Gulsen Colakoglu for their insightful critiques and encouragement when needed. Throughout my stay in Columbus I have been blessed by the presence of a group of very special and dear friends, whose support and love I will always cherish and treasure. You have made my journey profoundly enjoyable and memorable, sharing with me the good as well as the bad times. My thanks and gratitude go out to all of you, especially to Niraj, Alice, Erica, Nadine, Sleiman, Rami, Ihab, the lovely Nohal, my wonderful and inspirational friend Nesrine, my eternal friend Sarine, the beautiful Noura, and my second sister Zeina. vi Last but not least, this and everything I have and will accomplish, I owe to three people who have made me the person that I am: my father, whose every step has been a guiding light along the way; my mother, whose unconditional love inspires me to become a better person; and my talented, gentle and loving sister, Nadine. Your trust, belief, and unwavering support instill in me the strength and determination to carry on through all obstacles and hardship. To you, I am eternally grateful and indebted. vii VITA April 24, 1981………………………...…….Born- Mimess, Lebanon June 2001…………………………..………Bachelor of Science in Biology The American University of Beirut Beirut, Lebanon June, 2003…………………………………Master of Science in Biology The American University of Beirut Beirut, Lebanon September 2003-Present………………..PhD Candidate, Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Ohio, USA PUBLICATIONS Uchida A, Alami NH, Brown A. Tight functional coupling of kinein-1A and dynein motors in the bidirectional transport of neurofilaments. Mol Biol Cell. In press. Alami NH, Brown A. Myosin Va increases the efficiency of neurofilament transport by decreasing the duration of long-term pausing. J Neuroscience. 2009 May 20; 29(20):6625-34. viii FIELD OF STUDY Major Field: Molecular, Cellular and Developmental Biology ix TABLE OF CONTENTS Abstract ................................................................................................................ ii Acknowledgements ............................................................................................. vi Vita ..................................................................................................................... viii List of Tables ..................................................................................................... xiii List of Figures .................................................................................................... xiv Chapter 1: INTRODUCTION .................................................................................1 1.1. Neurofilaments............................................................................................1 1.1.1. General introduction .............................................................................1 1.1.2. Neurofilament organization and assembly ...........................................5 1.1.3. Neurofilament function .........................................................................8 1.2. Axonal transport........................................................................................13 1.2.1. A historical perspective ......................................................................13 1.2.2. Fast axonal transport..........................................................................15 1.2.3. Slow axonal transport.........................................................................16 1.2.4. Polymers vs monomers......................................................................18 1.2.5. Neurofilament phosphorylation and transport ....................................19 1.3. Neurofilaments and neurodegenerative disease ......................................22 1.3.1. Amyotrophic lateral sclerosis..............................................................23 1.3.2. Alzheimer’s disease ...........................................................................25 1.3.3. Parkinson’s disease ...........................................................................26