Microtubules

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, p150[Glued] holds the most responsibility for effective microtubule organization throughout the cell and the necessary anchoring at the centrosome. P150[Glued] 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 tiem ; 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.

Dynactin is a multisubunit protein complex that functions as a processivity cofactor to cytoplasmic dynein, assisting in vesicle transport and cell division. Independent of dynein,dynactin also serves to anchor microtubules to the centrosome. The functions of the majority of dynactin's subunits have been described to a certain degree ; however, the p24 subunit remains largely uncharacterized. Among the few things that are known about p24 are that it has a predicted molecular weight of about 20,822 Da, forms an a-helix, and binds directly to the p150[Glued] subunit. In order to explore its function further, we have performed shRNA-mediated knockdown, and fluorescent microscopy. We observe that microtubule disorganization is amplified due to the loss of p24. Our findings support the model that p24 serves as reinforcement to stabilize p150[Glued] at the centrosome.

The centrosome is a dynamic and highly active organelle within the cell. It plays a pivotal role in mitosis driving several of the physical changes that are taking place. The centrosome self-replicates before mitosis in order to set up two spindle poles on opposite sides of the cell. This leads to the creation of a mother and daughter centrosomes within a cell that have distinct components. This project will examine the recruitment of proteins to the centrosome as a cell progresses through the cell cycle. The proteins examined are (Sd(B-tubulin, (Sf(B-tubulin, Nek 2, Centrin2, p150Glued, EB-1, and dynein intermediate chain. In addition, chromosome arrangement was determined. By examining these proteins we hope to establish a logical order for the interactions of these proteins and their key contributions to cell cycle progression and completion, specifically dealing with the development of the mother and daughter centrosomes.

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.

Dynein is a motor protein responsible for microtubule-based minus-end directed trafficking in eukaryotic cells. Dynactin is a protein complex involved in mitosis, embryonic development, intracellular trafficking and anchoring microtubules at the centrosome. While dynactin is widely recognized to improve the array of cargo with which dynein can associate, there has been some dispute over whether dynactin, which binds both dynein and microtubules, improves the distance that dynein can travel processively in the act of cargo trafficking before it dissociates from its microtubule. In this study, we compare movement parameters of wild type dynein-based vesicle movements with movements in cells where expression of dynactin's microtubule binding subunit, p150glued, has been knocked down. We find that dynactin does act as a processivity factor for dynein by increasing the distance that dynein can travel smoothly in a single movement event, but does not increase dynein's velocity.