Formation

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.

The kinesin superfamily of microtubule motor proteins is subdivided into families based upon structure and function. KIF9 is the founding member of the Kinesin-9 family, which is a largely uncharacterized group of kinesins. It was originally identified by sequence homology to other kinesins. Subsequent studies have shown that KIF9 interacts with proteins involved in cell shape remodeling, cell migration and proper centrosomal positioning. We have examined KIF9 function in mammalian cells using shRNA-mediated knockdown and GFP-plasmid overexpression. By knocking dow KIF9 expression in these cells, we have seen several effects on normal cell cycle progression. Using various cell cycle markers, we have observed a decrease in the number of cells in late S phase. In addition, there is a marked increase in the number of cells in early mitosis in unexpected time intervals. We propose that KIF9 is required for proper cell progression, via a potentially novel checkpoint mechanism.

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.

Kinesin motors bind to microtubules and function in mitosis and intracellular transport depending on the position of the motor domain within the primary sequence (Hirokawa and Noda 2008). KIF9 has recently been shown to be involved in MTOC positioning and mitotic entry in Dictyostelium (Tikhonenko et al. 2009). To determine if a similar role for KIF9 exists in mammalian cells, we are using siRNA-mediated knockdown of KIF9 in COS-7 cells. Analysis of unsynchronized and cell-cycle synchronized cells treated with siRNA to KIF9 reveal that the transition from G2 to M phase is delayed and that mitotic progression is also affected. Additionally, our data indicates that spindle pole function during anaphase may be abnormal in cells treated with siRNA, suggesting a role for KIF9 during that stage.

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.

Telomerase is associated with telomere production and nDNA protection. However, studies by Santos et al. have demonstrated that human telomerase has a mitochondrial entry sequence and in the presence of hydrogen peroxide it has been found inside the mitochondrion and may cause mitochondrial DNA mutations. Saccharomyces cerevisiae contains telomerase, but it does not have the mitochondrial entry sequence. To determine if the presence of telomerase in the mitochondria can induce mutations an experiment was developed in which a mitochondrion entry sequence would be fused to the S. cerevisiae telomerase enzyme. This fusion could then be screened in S. cerevisiae with an ade2 mutation for a simple color assay of mitochondrial activity. To date, no successful transformant has been identified. The frequency of incorrect ligations has been recognized and may indicate that the desired fusion is lethal to E. coli cells.

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.

Dynactin is a multi-subunit protein complex that is the required cofactor for the cytoplasmic motor protein, dynein. Dynactin is critical for cytoplasmic activities such as vesicle transport, cytoskeletal organization, membrane organization and mitotic progression as it serves to increase processivity of dynein. The main subunit in focus for this study is the sidearm extension p150Glued. On the exposed end (N-terminus) lie two different microtubule binding motifs: CAP-Gly and Basic. CAP-Gly has a high binding affinity for microtubules, while Basic binds the microtubules quite weakly. It is at this location that dynactin is proposed to assist in the processivity of transporting cargo long distances in the cell, by providing additional support for the dynein molecule as it progresses along the microtubule. Analysis of the effects of overexpression of these motifs, alone or in tandem, suggested that there was no disruption of the dynein/dynactin interaction, but its ability to act in a processive manner had been perturbed. Effects on non-dynamic processes, such as Golgi localization and microtubule organization were generally weak, but effects were greater on y-tubulin organization. While this could suggest a defect in centrosome organization, we propose that it instead shows that cargo motility in the affected cells is defective, and that transport of centrosomal proteins to the centrosome is interrupted, suggesting a broader cargo-transport defect may be evident in these cells.