T cell effector functions depend on focused secretion. This is accomplished by secretory vesicle (SV) clustering around the microtubule organizing center (MTOC) and MTOC translocation to the specialized site of cell-cell contact - the immunological synapse (IS). The dynein molecular motor has been implicated in both processes. To investigate the roles of dynein and dynein-associated proteins we used Jurkat cells expressing fluorescent CTLA-4 for SV tracking and molecular traps targeting dynein subunits to show that dynein intermediate chain (DIC) and the light chain LC8 are needed for both SV clustering and MTOC translocation. We also found that immunostaining with different anti-DIC antibodies labeled different pools of dynein at the IS in activated Jurkat cells. To discern how dynein separately accomplishes both MTOC and SV activities we cloned DIC cDNAs from Jurkat cell mRNA and obtained two isoforms, DIC2B and DIC2C. However, both isoforms were concentrated around the MTOC and formed a ring-like structure at the IS. We also saw little difference in dynein-binding proteins that co-immunoprecipitated with each isoform. We then investigated the roles of the dynactin component p150Glued and Lis1 protein in MTOC translocation and SV clustering. Surprisingly, p150Glued was concentrated around the MTOC but was not present at the IS. SVs marked by CTLA-4 showed clustering defects while MTOC translocation was not significantly affected in p150Glued siRNA knockdown cells. On the other hand, Lis1 immunostaining labeled a ring at the IS where it mimicked the distribution of the dynein ring thought to be involved in MTOC translocation. MTOC translocation was potently blocked in Lis1 siRNA knockdown cells but dynein recruitment was only slightly disrupted and there was no visible effect on actin localization at the IS. Overexpression of Lis1 or expression of Lis1 deletion mutants interfered with MTOC translocation and interfered with dynein recruitment, while actin was still localized at the IS. However, studies of calcium flux in response to T cell receptor (TcR) stimulation showed that these mutant-expressing cells had deficiencies in cell signaling from the TcR. These results suggest that MTOC translocation and SV clustering are mediated by dynein but likely involve different dynein-binding proteins. / text

T cells play pivotal roles in the immune system and focused secretion of either cytokines or cytotoxic molecules toward its target is crucial for T cell functions. This directional secretion involves two critical steps: the movement of the microtubule organizing center (MTOC) up to the cell-cell contact site and the directed movement of secretory vesicles towards the MTOC. The minus end-directed microtubule motor protein dynein was previously shown in our studies and those of others to accumulate and anchor at the contact site where it then draws the MTOC up to the contact site. A variety of studies led to the suggestion that there were two functionally different pools of dynein in Jurkat cells, one a ring-like structure that pulled the MTOC to the contact site and the other one uniquely corresponding to the distribution of dynactin. This led to the hypothesis that the second pool of dynein drove vesicle transport. To address this possibility, we used siRNA to deplete the cell of dynactin. These studies showed that almost complete knockdown of dynactin (p150[superscript Glued]) had little effect on MTOC translocation but it also had little effect on a panel of Golgi vesicle markers, whose movement the literature suggested was dynein dependent. As an alternative, a Jurkat cell line expressing fluorescent CTLA4, a known marker for the secretory lysosomes was generated. CTLA4 accumulated at the contact site when Jurkat cells made contact with synthetic target cells. When we repeated the p150[superscript Glued] knockdown in these cells, we found that vesicle transport was blocked, whereas MTOC polarization remained normal. These studies suggest that dynein serves critical roles in both aspects of T cell effector function, the movement of the MTOC up to the cell-cell contact site and the movement of a special class of secretory vesicles up to the MTOC. By the combined processes of MTOC translocation and the minus end-directed movement of vesicles, T cells make it so that a concentrated pool of secretory vesicles are aimed to secrete locally only towards target cells. This ensures that the antigen-specificity of T cell activation is followed by a localized response aimed at the intended target cell. / text

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial cells form the lining of the walls of blood vessels and are continuously subjected to mechanical stimuli from the blood flow. Microtubule-organizing center (MTOC), also known as centrosome is a structure found in eukaryotic cells close to the nucleus. MTOC relocates relative to the nucleus when endothelial cells are exposed to shear stress which determines their polarization, thus it plays a critical role in cell migration and wound healing. The nuclear lamina, a mesh-like network that lies underneath the nuclear membrane, is composed of lamins, type V intermediate filament proteins. Mutations in LMNA gene that encodes A-type lamins cause the production of a mutant form of lamin A called progerin and leads to a rare premature aging disease known as Hutchinson-Gilford Progeria Syndrome (HGPS). The goal of this study is to investigate how fluid flow affects the cytoskeleton of endothelial cells. This thesis consists of two main sections; computational mechanical modeling and laboratory experimental work. The mechanical model was implemented using Ansys Workbench software as a tensegrity-based cellular model in order to simulate the state of an endothelial cell under the effects of induced shear stress from the blood fluid flow. This tensegrity-based cellular model - composed of a plasma membrane, cytoplasm, nucleus, microtubules, and actin filaments - aims to understand the effects of the fluid flow on the mechanics of the cytoskeleton. In addition, the laboratory experiments conducted in this study examined the MTOC-nuclear orientation of endothelial cells under shear stress with the presence of wound healing. Wild-type lamin A and progerin-expressing BAECs were studied under static and sheared conditions. Moreover, a custom MATLAB code was utilized to measure the MTOC-nuclear orientation angle and classification. Results demonstrate that shear stress leads to different responses of the MTOC orientation between the wild-type and progerin-expressing cells around the vertical wound edge. Future directions for this study involve additional experimental work together with the improved simulation results to confirm the MTOC orientation relative to the nucleus under shear stress.