CONTRIBUTIONS of DYNACTIN's P150glued SUBUNIT's BINDING

CONTRIBUTIONS OF DYNACTIN’S p150Glued SUBUNIT’S BINDING DOMAINS TO MICROTUBULE ANCHORING AT THE CENTROSOME by Rebecca Schneider A Thesis Submitted to the Faculty of The Harriet L. 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 Biological Chemistry Harriet L. Wilkes Honors College of Florida Atlantic University Jupiter, Florida May 2012 CONTRIBUTIONS OF DYNACTIN’S p150Glued SUBUNIT’S BINDING DOMAINS TO MICROTUBULE ANCHORING AT THE CENTROSOME by Rebecca Schneider This thesis was prepared under the direction of the candidate’s thesis advisor, Dr. Nicholas Quintyne, and has been approved by the members of her/his 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. Christopher Strain ______________________________ Dean, Wilkes Honors College ____________ Date ii Acknowledgements Dr. Quintyne, for mentorship, leadership, and patience. April, for advice, skittles, and thermometers. Dr. Strain, for perspective. Dr. King, for supplies and ideas. Megan, Emma, & Rachel, for putting up with science talk for four years. Lab-mates for learning alongside me. My family & friends for love and support, always. iii for all the people without whose support I could not have accomplished this iv Abstract Author: Rebecca Schneider Title: Contributions of dynactin’s p150Glued subunit’s binding domains to microtubule anchoring at the centrosome Institution: Harriet L. Wilkes Honors College of Florida Atlantic University Thesis Advisor: Dr. Nicholas Quintyne Concentration: Biological Chemistry Year: 2012 Intracellular transport carries out very important roles within the cell including mitosis, organization, and organelle function. In order for effective transport using the motor protein dynein a cofactor named dynactin is required. Of dynactin’s many subunits p150Glued holds the most responsibility for effective microtubule organization throughout the cell and the necessary anchoring at the centrosome. P150Glued holds two areas of high binding potential, the CAP-Gly region and the Basic region. Each of these binding domains have different binding potentials and affinities for microtubules. The CAP-Gly region binds tightly the microtubules for a longer period of time; the Basic region binds loosely to microtubules. Throughout the course of my research I manipulated these two regions binding affinity for microtubules and evaluated the resulting cells ability to effectively organize microtubules and anchor them properly at the centrosome. v Table of Contents Introduction………………………………………………………...………………..Page 1 Methods……………………………………………………………...………………Page 7 Cell Culture…………………………………………...……………..Page 7 Plasmids……………………………………………..………………Page 7 Antibodies……………………………………………...……………Page 7 Lipid Transfections……………………………………...…………..Page 7 Selection of shRNA…………………………………………………Page 8 Immunofluorescence…………………………………...……………Page 8 Mitotic Index…………………………………………...……………Page 9 Results………………………………………………………………….…………..Page 10 Discussion……………………………………………………………….…………Page 24 Works Cited………………………………………………………………………..Page 28 vi Figures and Tables Motor Proteins (Figure 1)……………………………………………………Page 1 Dynactin Structure (Figure 2)……………………………………………….Page 2 Dynein & Dynactin Interactions (Figure 3)……………….………...………Page 3 p150Glued Structure (Figure 4)………………………………………….……Page 4 Microtubule Anchoring (Figure 5)…………………………….…………….Page 5 Microtubule Organization (Figure 6)…………………………………...….Page 11 Golgi Localization (Figure 7)…………………………...………………….Page 13 p150Glued at the Centrosome (Figure 8)…………………...………………..Page 15 γ-tubulin Foci at the Centrosome (Figure 9)……………………………….Page 17 Microtubule Organization (Figure 10)………………………………..……Page 19 γ-tubulin Foci at the Centrosome (Figure 11)………………………...……Page 21 Microtubule Organization (Figure 12)…………………………………..…Page 22 γ-tubulin Foci at the Centrosome (Figure 13)…………………………...…Page 23 vii Introduction Within the cell, cytoskeletal architecture serves many integral functions. These functions range from providing support for cell shape, to facilitating cell division (Dogterom and Yourke, 1977). The cytoskeleton has several components, among these are the actin-based microfilaments (MF’s), the intermediate filaments (IF’s) and the microtubules (MT’s). Effective cellular trafficking is facilitated by cellular motor proteins, such as the myosin and kinesin families as well as cytoplasmic dynein (Hirokawa, 1998). These motor proteins move along the cytoskeleton in fixed direction transporting cargo around the cell using active transport by hydrolyzing ATP into ADP to facilitate their travel. Microtubule motor proteins such as dynein and kinesin are used for effective transport of cargo around the cell. These motor proteins have unique structures displayed in Figure 1. Figure 1: Diagrams of Kinesin motors (left) and dynein motors (right). The figure on the left shows kinesin’s two heads (yellow), stalks (white), and tails (green). The figure on the right shows dynein’s heavy chains (purple), intermediate chains (green), light intermediate chains (red) and 1 light chains (yellow). Transport using dynein requires a cofactor called dynactin for efficient transport. (Gill et al., 1991). Dynactin is a multi-subunit protein whose association with dynein increases its processivity and effectiveness for intracellular movement (Schroer & Sheetz, 1991). Dynactin’s multi-subunit structure is displayed in Figure 2. Figure 2: Model of Dynactin's subunits. This diagram was developed ___. From this diagram the current breakup of regions was developed with the Shoulder/Sidearm Glued subunit composed of p150 , p24, and dynamitin. And the Arp minifilament backbone being composed of the rest of the subunits. The many subunits of dynactin can be divided in to three groups; the microtubule binding subunits, the motor binding subunits, and the cargo binding subunits. In the dynactin structure, Arp1, Arp11, Actin, p62, p27, p25 and CapZ act with various cargo binding capacities (Schroer & Sheetz, 1991: Eckley et al., 1999). Dynein itself binds to the p150Glued structure via the stalk that extends upwards from the complex (Boylan et al., 2000; Vaughan et al., 2002; King, 2000). The shoulder/sidearm area, composed of p150Glued, p24 and dynamitin is responsible for binding to the microtubules (Waterman- Storer et al., 2995; Eckley et al., 1999; Quintyne et al., 1999; Culver-Hanlon et al., 2006). The microtubule binding domains are located on the two globular heads of the p150Glued 2 subunit. The interactions between the cargo, dynactin, and dynein as they pertain to effective transport are cartooned in Figure 3. Figure 3:Dynactin and dynein interactions between various subunits and points of contact. (Schliwa et al., 2003) As far as dynactin is concerned, microtubule binding is one of its most important functions. The p150Glued subunit carries in the responsibility of motor binding and enchancement of motor processivity (Schroer & Sheetz, 1991). A dynactin molecule contains a homodimer of p150Glued, the stalks arranged in an α-helical coiled coil (Quintyne et al., 1999), and at the end, two heads with high microtubule affinity. These two areas have been labeled the CAP-Gly Binding Domain, and the Basic Binding Domain (Culver-Hanlon et al, 2006). These two binding domains’ placement on the secondary structure of the protein is diagrammed out in Figure 4 (Culver-Hanlon et al., 2006). 3 Figure 4: Components of Dynactin’s p150Glued Subunit. This diagram shows the entire length of the subunit from 1-1325. On the overall dynactin picture, the green and red regions are visible on the globular heads of p150Glued. The purple represents to the stalk regions of p150Glued. The two binding domains of p150Glued are the main reason that dynactin is so effective at helping dynein increase intracellular trafficking. These binding domains serve different functions for the dynactin complex as they pertain to microtubule binding. These two areas of high affinity are characterized by very different biochemical makeups and affinities for their targets. The CAP-Gly binding domain has been observed to have a high affinity for microtubules and tends to continue its affinity to a specific point for long periods of time, possibly acting as an “anchor or parking brake”. The more recently discovered basic binding domain is characterized by a large number of basic amino acids and is believed to exert a looser grip on the microtubules (Culver-Hanlon et al., 2006). 4 These two binding domains various interactions with the microtubules allow for the dynactin complex as a whole to increase dynein processivity along microtubules. Aside from assisting in effective transport of cargo around the cell, another one of dynactin’s major functions is to anchor microtubules at the centrosome (Quintyne et al., 1999). As the microtubule organizing center (MTOC) of the cell, the centrosome is the cornerstone of effective microtubule use through accurate organization. There is a complex of proteins that assists in the effective anchoring of microtubules to the centrosome (Quintyne et al., 1999: Mogensen et al., 2000) . If this anchoring is disrupted, the dynamic microtubules have no starting and ending point and their ability to build and rebuild themselves effectively

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