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- Title
- You keep me hangin' on: dynactin's p24 is essential for microtube anchoring.
- Creator
- Le, Ariel., Harriet L. Wilkes Honors College
- Abstract/Description
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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...
Show moreDynactin 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.
Show less - Date Issued
- 2012
- PURL
- http://purl.flvc.org/FAU/3355592
- Subject Headings
- Cytoskeletal proteins, Cell organellles, Formation, Microtubules, Molecular biology
- Format
- Document (PDF)
- Title
- Knockdown of dynactin's p150[Glued] subunit abrogates microtubule organization.
- Creator
- Roeckner, Jared Todd., Harriet L. Wilkes Honors College
- Abstract/Description
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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...
Show moreDynactin 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.
Show less - Date Issued
- 2009
- PURL
- http://purl.flvc.org/FAU/209998
- Subject Headings
- Cytoskeletal proteins, Cell organelles, Formation, Microtubules, Molecular biology
- Format
- Document (PDF)
- Title
- Investigation of talin head-tail interactions.
- Creator
- Butyn, Amber., Harriet L. Wilkes Honors College
- Abstract/Description
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Talin is a ubiquitous, high-molecular-weight, flexible protein that plays a critical role in focal adhesions by activating, as well as connecting, integrins to the actin cytoskeleton. Talin's inactive auto-inhibitory state is speculated to be one of its modes of regulation inside the cell and is achieved through its head-tail interactions. In order to decipher the stability of this interaction, the head domain (residues 206-405) was cloned into a modified pET28m vector while the tail domains ...
Show moreTalin is a ubiquitous, high-molecular-weight, flexible protein that plays a critical role in focal adhesions by activating, as well as connecting, integrins to the actin cytoskeleton. Talin's inactive auto-inhibitory state is speculated to be one of its modes of regulation inside the cell and is achieved through its head-tail interactions. In order to decipher the stability of this interaction, the head domain (residues 206-405) was cloned into a modified pET28m vector while the tail domains (residues 1654-2344 and 2225-2344) were cloned into the pET32a vector to obtain octa-histidine tagged and un-tagged peptide, respectively. Neither co-expression nor pull-down using the His-tagged head domain was successful in purifying a stable head-tail complex. Our results indicate rather weak interactions between the talin head and rod domains and hence, under our experimental conditions, do not lead to a stable auto-inhibitory complex that can be purified for further studies.
Show less - Date Issued
- 2009
- PURL
- http://purl.flvc.org/FAU/209984
- Subject Headings
- Membrane proteins, Structure-activity relationships, Cell membranes, Physiology, Molecular biology, Cell interaction, Developmental cytology
- Format
- Document (PDF)
- Title
- FSTL-1 secreted by mesenchymal stem cells increases cell viability of human aortic endothelial cells under hypoxic stress.
- Creator
- Eid, Nibal., Harriet L. Wilkes Honors College
- Abstract/Description
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Human mesenchymal stem cells (MSCs) are being evaluated for the treatment of a broad array of diseases due to their ability to secrete a variety of therapeutically beneficial paracrine-acting factors. For example, MSC conditioned media (MSC-CM) has been shown to inhibit hypoxia-induced apoptosis in human aortic endothelial cells (HAECs) via activation of the P13-AKT pathway. However, the factors secreted by MSCs responsible for this effect have yet to be identified. Recent studies have shown...
Show moreHuman mesenchymal stem cells (MSCs) are being evaluated for the treatment of a broad array of diseases due to their ability to secrete a variety of therapeutically beneficial paracrine-acting factors. For example, MSC conditioned media (MSC-CM) has been shown to inhibit hypoxia-induced apoptosis in human aortic endothelial cells (HAECs) via activation of the P13-AKT pathway. However, the factors secreted by MSCs responsible for this effect have yet to be identified. Recent studies have shown that the glycoprotein Follistatin-like 1 (FSTL1) activates the P13-AKT pathway by binding to the receptor disco-interacting protein (DIP2A) expressed on the surface of cells. Based on our data indicating that MSCs constitutively secrete high quantities of FSTL1, we hypothesize that this protein principally mediates the anti-apoptopic effect of MSC-CM on HAECs. Loss-of-function studies employing siRNA-mediated knockdown of the protein and neutralizing antibodies will be used to assess the role of FSTL1 in growth and survival of HAECs following exposure to hypoxic stress.
Show less - Date Issued
- 2012
- PURL
- http://purl.flvc.org/FAU/3359296
- Subject Headings
- Stem cells, Transplantation, Molecular biology, Gene therapy, Coronary heart disease, Prevention, Stress (Physiology)
- Format
- Document (PDF)
- Title
- Dynactin is a processivity factor for dynein in vivo.
- Creator
- Fulton, Edward., Harriet L. Wilkes Honors College
- Abstract/Description
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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...
Show moreDynein 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.
Show less - Date Issued
- 2009
- PURL
- http://purl.flvc.org/FAU/209990
- Subject Headings
- Cell organelles, Formation, Microtubules, Cytoskeletal proteins, Molecular biology
- Format
- Document (PDF)
- Title
- Contributions of dynactin's p150[Glued] subunit's binding domains to microtubule anchoring at the centrosome.
- Creator
- Schneider, Rebecca., Harriet L. Wilkes Honors College
- Abstract/Description
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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...
Show moreIntracellular 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.
Show less - Date Issued
- 2012
- PURL
- http://purl.flvc.org/FAU/3359326
- Subject Headings
- Cytoskeletal proteins, Microtubules, Centrosomes, Cell organelles, Formation, Molecular biology, Tubulins
- Format
- Document (PDF)