Knockdown of dynactin’s p150Glued subunit abrogates microtubule organization by Jared Todd Roeckner A Thesis Submitted to the Faculty of The Wilkes Honors College In Partial Fulfillment of the Requirements for the Degree of Bachelor of Arts in Liberal Arts and Sciences with a Concentration in Biology Wilkes Honors College of Florida Atlantic University Jupiter, Florida May 2009 Knockdown of dynactin’s p150Glued subunit abrogates microtubule organization by Jared T. Roeckner This thesis was prepared under the direction of the candidate’s thesis advisor, Dr. Nicholas Quintyne, and has been approved by the members of the supervisory committee. It was submitted to the faculty of The Honors College and was accepted in partial fulfillment of the Requirements for the Degree of Bachelor of Arts in Liberal Arts and Sciences. SUPERVISORY COMMITTEE: ________________________ Dr. Nicholas Quintyne ________________________ Dr. Paul Kirchman ________________________ Dean, Wilkes Honors College _________ Date ii ACKNOWLEDGEMENTS First, I would like to thank Dr. Nicholas Quintyne for allowing me to work in his lab for the last three years and overseeing and guiding my thesis research and writing. I would like to acknowledge Dr. Stephen King and Dr. Margret Kincaid at UMKC for providing us with the p150Glued knockdown plasmids. Dr. Paul Kirchman, April Mistrik, and everyone in the Quintyne lab helped me out greatly. Ed Fulton and I worked on many steps of this project together and I thank him for his help. Finally, I would like to thank my family and friends for supporting of my thesis research and my undergraduate studies as a whole. iii ABSTRACT Author: Jared Todd Roeckner Title: Knockdown of dynactin’s p150Glued subunit abrogates microtubule organization Institutions: Harriet L. Wilkes Honors College, Florida Atlantic University Thesis Advisor: Dr. Nicholas Quintyne Degree: Bachelors in Liberal Arts and Sciences Concentration: Biology Year: 2008-2009 Dynactin is a multifunctional protein complex composed of at least 11 different subunits. Dynactin functions as a cofactor for cytoplasmic dynein facilitating long-range vesicle movements, microtubule anchoring, endomembrane localization, and mitotic progression. Previous studies have shown that dynactin binds to microtubules at the centrosome maintaining a radial array in interphase. The p150Glued subunit contains two distinct microtubule-binding sequences named CAP-Gly and Basic. While both domains can interact with microtubule, CAP-Gly has a much greater affinity for binding to microtubules, suggesting that the two domains may be active for different dynactin-based functions within the cell. Using siRNA, we found that knockdown of p150Glued was sufficient to alter the maintenance of radial microtubule arrays, cause an increase in centrosome number and mitotic index. In the future we will replace the endogenous protein with versions lacking the CAP-Gly or Basic domains to investigate the contribution of each to microtubule anchoring and cytoskeletal architecture. iv TABLE OF CONTENTS INTRODUCTION 1 METHODS 10 Cell Culture 10 Plasmid Preparation 10 Transfection 11 Immunofluorescence 11 Immunoblotting 12 RESULTS 13 Purification of plasmid 13 Western blotting suggests partial p150Glued knockdown 13 Determination of transfected cells 14 Loss of radial array 15 Loss of p150Glued increases centrosome number 16 Loss of p150Glued increases mitotic index 17 DISCUSSION 18 REFERENCES 21 v FIGURES and TABLES Figure 1. Structural model of cytoplasmic dynein 2 Figure 2. Dynactin Ultrastructural Model 4 Figure 3. Schematic of dynactin p150Glued 6 Figure 4. Proposed model of centrosomal anchoring 8 Figure 5. DNA gel 13 Figure 6. Expression of p150Glued 14 Figure 7. Representative images of co-transfected cells 15 Figure 8. Radial Array 16 Figure 9. Increase in supernumerary centrosomes 17 Figure 10. Increase in mitotic index 18 Figure 11. Representative image of a binucleate cell 18 vi INTRODUCTION Dynactin was first identified as a cytosolic activity that increased cytoplasmic dynein processivity along microtubules (Schroer & Sheetz, 1991). Further work determined that dynactin was a multisubunit protein consisting of at least 11 domains (Gill et al., 1991; for a review see Schroer, 2004). It is important in many cellular processes. Dynactin is required for mitosis in multicellular organisms and plays a role in subcellular movement and cargo binding (Echeverri et al., 1996; Burkhardt et al., 1997; Valetti et al., 1999). Dynactin is a required factor for microtubule anchoring at the centrosome (Quintyne et al., 1999). Disruption of dynactin leads to a breakdown of plus- end directed and minus-end directed organelle transport (Deacon et al., 2003). Research into the function and regulation of dynactin has lead to insights in the mechanics of degenerate neurons (Lane et al., 2001; Puls et al., 2003). A subunit of dynactin, p150Glued, may be involved in muscular dystrophy (Tokito & Holzbaur, 1998). Cytoplasmic Dynein Cytoplasmic dynein is a minus-end directed microtubule motor (Paschal & Vallee, 1987) thought to be involved in vesicle movement, spindle pole retention, organelle retention, and chromosome movement (for a review see Holzbaur & Vallee, 1994). Cytoplasmic dynein was first identified in sea urchin eggs, (Hisanaga & Sakai, 1983) then in Caenorhabditis elegans (Lye et al., 1987) and bovine brain (Paschal et al., 1987). Antibodies to dynein subunits have been shown to localize at the kinetochores during mitosis (Pfarr et al., 1990). Dynein has also been found to associate with various membrane organelles such as endocytic vesicles, lysosomes, microsomes, and parts of the 1 Goli apparatus (Lin and Collins, 1992; Corthezy-Theulaz et al., 1992). As shown in figure 1, dynein is a multisubunit protein that consists of two catalytic heavy chains (HC, 532 kDa), several intermediate chains (IC, 74kDa), light intermediate chains (LIC 53- 59kDa), and light chains (LC) (Paschal et al., 1987; Vaughan & Vallee, 1995; King & Schroer, 2000). By using energy from ATP, the heavy chains generate force to move the protein and cargo along microtubules. The LC are involved in cargo binding, the LIC binds directly to the dynein heavy chain and may mediate the attachment of dynein to centrosomes and other cargoes, and IC are the location of dynactin binding. Heavy Chains Light Intermediate Chains Light Chains Intermediate Chains Figure 1. Structural model of cytoplasmic dynein. (Figure courtesy of SJ King). Dynactin Dynactin (shown in Figure 2) is multisubunit protein complex (Gill et al., 1991) that allows cytoplasmic dynein to carry out long-range vesicle movements on microtubules in vitro (Schroer & Sheetz, 1991). Studies in yeast, Drosophila, and filamentous fungi firmly established dynactin as a necessary cofactor to cytoplasmic dynein (for a review 2 see Schroer, 1994). Because dynactin is required for mitosis in multicellular organisms, it is essential for viability. It has been shown that mutations to dynactin subunits can cause defects in axonal transport (Puls et al., 2003). The largest subunit of dynactin, p150Glued, participates in binding to dynein and increased motor processivity (Karki & Holzbaur, 1995; Vaughan & Vallee, 1995; King & Schroer, 2000). The subunit p150Glued is also a target of apoptotic cleavage and therefore is important for normal cell function (Lane et al., 2001). Structure and Subunits Protein isolation from animal brains has allowed for the study and characterization of dynactin (Bingham et al., 1998). Dynactin is complex molecule composed of 11 distinct peptide units that make up a projection arm and rod-like domain. Since there are multiple copies of certain subunits, each dynactin molecule is made of over 20 individual peptides, has a mass of approximately 1.2 MDa (Schroer, 2004) and is divided into two distinct domains. Most of the mass is contained in the projecting arm (also called the shoulder/sidearm), which includes p150Glued, dynamitin, and p24/p22 (Schroer, 2004). The shoulder/sidearm allows for microtubule attachment. The rod-like domain includes CapZ, Arp1, Arp11 and other proteins, and serves to allow dynactin to bind to various membranes and intracellular cargos. 3 Motor Binding MT binding of p150Glued p22/p24 dynamitin Cap Z Arp1 Arp11 Cargo Binding Figure 2. Dynactin Ultrastructural Model. Dynactin is a multisubunit protein complex that has been shown to be a required cofactor of dynein and is also required for MT anchoring at the centrosome. The two globular heads of the long arm, p150glued, have MT binding sites. (Figure adapted from Quintyne et al., 1999). Arp1 Rod The Arp1 rod is approximately 10 x 40nm, resembles an actin filament, and is composed of Arp1, Arp11, Actin, CapZ, p62, p27, and p25. Arp1 can hydrolyze ATP (Bingham & Schroer, 1999) and bind directly to Golgi structures via spectrin family proteins (Holleran et al., 2001). The end of Arp1 terminates with CapZ, a plus end actin-capping protein (Schafer et al., 1994). Arp11 caps the minus end of Arp1. The p62 subunit is thought to be involved in protein-protein binding because it has a zinc-binding motif (Eckley et al., 1999). p25 and p27 may serve as adapter proteins allowing the Arp1 rod to bind to various subcellular structures (Eckley et al., 1999). 4 The Shoulder/sidearm The shoulder/sidearm extends from the Arp1 rod and contains three subunits, p150Glued, dynamitin, and p24/p22. Dynactin molecules have two copies of p150Glued, two copies of p24/p22, and four copies of dynamitin which self-associate