Browsing by Subject "Cytoskeleton"
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Item Comparative analysis of the molecular pathways involved in spinal cor injury in axolotls vs. mammaals(2015-04) Diaz Quiroz, Juan FelipeSpinal cord injuries (SCI) in mammals are major causes of physical disabilities. In contrast the Mexican salamander (axolotl) possesses amazing regenerative capabilities and can regenerate a fully functional spinal cord after injury. We have attempted to identify the molecular determinants underlying this evolutionary divergence by undertaking a detailed comparative analysis of a regenerative model, the axolotl spinal cord, and a corresponding non-regenerative system, rat spinal cord after injury. This approach identified a small number of highly conserved microRNAs that are differentially regulated in axolotl versus. Detailed in vivo studies of one of these microRNAs, miR-125b that is highly expressed in axolotl but low in rat has identified it as a key regulator of the regenerative response in axolotl. We also found that upregulation of miR-125b after injury improves SC repair in rats. In addition, we have identified SEMA4D as a target gene regulated by this microRNA after SCI in both axolotl and rat. We also studied how this miRNA is regulated in the axolotl to promote regeneration after SCI. We found that in the absence of injury, actin cytoskeleton de-polymerization induced by Cytochalasin D (Cyto D) induced a decrease in miR-125b expression similar to the decrease induced by injury in axolotl. Analyses of the regulatory region of the miR-125b axolotl gene revealed predicted binding sites for c-Fos. When we performed SCI in axolotls, we observed an increase in the expression of c-Fos 1 day after injury. As expected, we found that in the absence of injury, treatment with Cyto D induced similar changes in c-Fos expression similar to injury. Lastly, we proposed the development of a 3D in vitro co-culture model system to study at the cellular and molecular level how miR-125b SCI regulates the response to injury in rats and whether miR-125b expression is regulated by biomechanical activation of the Rho-c-Fos pathway. The proposed model will allow better imaging, better spatial and temporal control over modulating microRNA and gene expression in different cells at different time points. Our overall data suggest that dynamic changes in the actin cytoskeleton after injury induces changes in miR-125b expression, possibly through activation of cFos in the RhoA pathway, to create a permissive environment for regeneration in axolotls.Item Mechanisms and models of dehydration and slow freezing damage to cell membranes(2010-10) Ragoonanan, VishardCell preservation is accomplished primarily by two methods: cryopreservation and dehydration, with the former being the standard technique used. In order to optimize and develop cell preservation protocols for cells that are difficult to preserve or whose end application is incompatible with current cell preservation protocls and to advance preservation by dehydration, a better understanding of the freeze- and dehydration-induced changes to the cell membrane is required. Despite a large body of literature on the topic, the mechanisms of damage to cells during slow freezing and dehydration are still ambiguous. The objective of this study is to investigate the mechanisms of damage to the cell membrane during slow freezing and dehydration and expand our outlook beyond the cell membrane to its underlying support, the cytoskeleton. In this study, we used several model systems to investigate slow freezing and dehydration. We used a liposome model to gather basic information on changes that can occur to a simple membrane system during freezing. This study revealed that eutectic formation was capable of dehydrating the membrane at low temperatures which may be contribute to alteration of the post-thaw membrane structure. We used a bacteria model to investigate the role of the phase transition and immediate versus slow osmotic stress on post-rehydration viability. This study revealed that going through a lyotropic membrane phase transition was detrimental to post-rehydration viability. This study also demonstrated that a rapidly applied osmotic stress was more detrimental to the structure/ organization of the membrane than gradual osmotic stress. We then subjected a model mammalian cell to both hyperosmotic stress and freeze-thaw and investigated both the membrane and cytoskeletal responses. Osmotic stress experiments suggested that alterations in membrane structure (i.e., surface defects and lipid dissolution) were directly dependent on the change in the chemical potential of water. These experiments also suggest that cell shrinkage and the resulting formation of membrane protrusions negatively affect viability upon return to isotonic conditions. It was found that membrane morphology in the dehydrated state and post-hyperosmotic viability was dependent on the stiffness of the cytoskeleton. Freeze/ thaw experiments suggested that ice-cell interaction decreases post-thaw viability. However, similar to osmotic stress experiments, cell shrinkage and cytoskeletal stiffness negatively impact post-thaw viability. We suggest the resulting membrane morphology due to cell shrinkage is also responsible for damage during freeze/ thaw. The various mechanisms discovered and the models proposed can be used in developing new protocols for cell preservation and for cell destruction (e.g. cryosurgery).Item Optimality in the nanomechanics of cell migration and adhesion(2014-09) Bangasser, BenjaminCell migration is key to many biological processes including embryonic development, wound healing, and disease progression, and the mechanical stiffness of a cell's environment exerts a strong, but variable, influence on this migration. Many cells display a stiffness optimum at which migration is maximal, however, these stiffness optima span several orders of magnitude, from ~1-1000 kPa, suggesting that different cell types possess distinct operating parameters. Firstly, we describe how a motor-clutch model of cell traction, which exhibits a maximum in traction force with respect to substrate stiffness, may provide a mechanistic basis for understanding how cells are "tuned" to sense the stiffness of specific microenvironments. We found that the optimal stiffness is generally more sensitive to clutch parameters than to motor parameters, but that single parameter changes are generally only effective over a small range of values. By contrast, dual parameter changes, such as coordinately increasing the numbers of both motors and clutches, offer a larger dynamic range for tuning the optimum. The model exhibits distinct regimes with "frictional slippage" at both low and high substrate stiffness where clutches are inefficiently utilized. Between the two extremes, we find the maximum traction force where clutches are most efficiently utilized, which occurs when the substrate load-and-fail cycle time equals the expected time for all clutches to bind. Secondly, we also present a master equation-based ordinary differential equation (ODE) description of the motor-clutch model, from which we derive an analytical expression for a cell's optimum stiffness. This analytical expression provides insight into the requirements for stiffness sensing by establishing fundamental relationships between the key controlling cell-specific parameters. Both the ODE solution and the analytical expression show good agreement with Monte Carlo motor-clutch output, and reduce computation time by several orders of magnitude, which potentially enables long time scale behaviors (hours-days) to be studied computationally in an efficient manner. The ODE solution and the analytical expression may be incorporated into larger scale models of cellular behavior to bridge the gap from molecular time scales to cellular and tissue time scales. Thirdly, to create a unified theoretical framework for cell migration, we have developed and experimentally tested a whole cell migration simulator based on the motor-clutch model by imposing coupled force balances and mass balances on molecular motors, adhesion molecules ("clutches"), and actin subunits in a compliant microenvironment. The model predicts a stiffness optimum that can be shifted by altering the number of active molecular motors and clutches. This prediction was verified experimentally by comparing cell traction and F-actin retrograde flow for two cell types with differing amounts of active motors and clutches: embryonic chick forebrain neurons (ECFNs; optimum ~1 kPa) and U251 glioma cells (optimum ~1000 kPa). In addition, the model predicted, and experiments confirmed, that the stiffness optimum of U251 glioma cell migration, projected area, aspect ratio, F-actin flow rate, and traction strain energy can be shifted to lower stiffness by simultaneous drug inhibition of myosin II motors and integrin-mediated adhesions. Overall, the motor-clutch cell migration simulator provides a unified theoretical framework with which to predict cell adhesion and migration in defined mechanochemical environments.Item An unconventional myosin is necessary for chemotaxis in Dictyostellium discoideum.(2009-08) Breshears, Laura MarieDirected cell migration (chemotaxis) is a fundamental biological process necessary for embryonic development, wound healing, and proper function of the immune system. Chemotaxis also plays a significant role in many developmental disorders and post-embryonic diseases in humans, such as cancer. Chemotaxis is driven by extracellular cues that act, in large part, to induce changes in the actin cytoskeleton, such as actin polymerization, that facilitate directed cell migration. Myosins are actin-associated motors that have a variety of functions in different cellular contexts. Myosins can effect cortical tension, pseudopod and filopodia formation, phagocytosis, the function of sensory structures, and the basic mechanics of cell motility. Members of the MyTH/FERM family of unconventional myosins all have roles in actin-based processes and one member, vertebrate myosin X, has recently been shown to play a role in actin dynamics in response to extracellular migration cues. The social amoeba Dictyostelium discoideum is a powerful model system for dissecting chemoattractant signaling pathways and identifying the cytoskeletal components necessary for directed cell migration. MyoG is a novel unconventional myosin characterized by two MyTH/FERM domains in its tail region. The potential role of this myosin in Dictyostelium cell migration was investigated by analyzing the phenotype of three independent myoG null mutants. The initial stages of Dictyostelium development, induced by starvation, depend on chemotaxis to cAMP, resulting in the formation of a multi-cellular aggregate. Upon starvation myoG — cells fail to aggregate, arresting as a smooth monolayer of cells. The myoG — cells neither polarize in a cAMP gradient nor do they chemotax toward the cAMP source. Analysis of the ability of myoG — cells to polymerize actin in response to cAMP revealed that the response is dampened in the mutants. myoG — cells are also defective in signaling to PI3K in response to cAMP. These data show that while the mutant cells retain some ability to respond to the gradient, the major pathways regulating polarity and chemotaxis are not functional. The mutant phenotype suggests that MyoG acts in transducing the chemotactic signal from the cAMP receptor to PI3K and the actin cytoskeleton, facilitating the morphological changes that lead to polarization and directional migration. The role of MyoG in chemotactic signaling represents a novel function for an unconventional myosin. The work presented here clearly demonstrates that MyoG is necessary for signaling from the cAMP receptor to both PI3K and the actin cytoskeleton. Sequence analysis shows that there is no direct homologue of MyoG in other organisms, but the high degree of conservation of the chemotactic signaling pathways indicates that there are likely to be functional homologues in higher eukaryotic cells, such as neutrophils, that rely on chemotaxis for cellular function.