Cardiovascular disease is the leading cause of human death worldwide. Currently, the prevalence of cardiovascular disease and health care costs associated with its onset continue to increase in both developed and developing societies. Concordant with the need to improve preventative measures is the imperative to develop more effective and efficient remedies for incident cardiovascular pathologies. Increased aortic stiffness with aging has recently emerged as an early, independent, and consistent physiological predictor of cardiovascular disease and represents an attractive target for possible therapeutic options. The success of any biomedical strategy in this regard is incumbent upon comprehension of biological processes and mechanical properties attributable to constituent components within the aortic wall. This dissertation tested the hypothesis that aging-induced changes to smooth muscle maintenance of biomechanical homeostasis within the aorta lead to undesirable increases in stiffness, correlative with increased risk of negative cardiovascular outcomes. Conventionally, mechanical studies and models have identified extracellular matrix as the primary determinant of changes in stiffness, but new research presented here shows that this may not be true. In viable ex vivo preparations of aortic tissue, roughly half of the maximal elastic modulus results from alpha-agonist activation of smooth muscle cells. Investigation of the biochemical interactions that characterize this effect revealed a link between aging and decreased expression of Src, a kinase involved in numerous signaling pathways governing cellular growth and survival, as well as defective regulation of focal adhesions between the smooth muscle cells and extracellular matrix. These findings were integrated into a model of aortic contractility and stiffness that establishes an aging-impaired regulatory complex comprising focal adhesions and non-muscle actin cytoskeleton in vascular smooth muscle cells. A better understanding of the mechanisms underlying this model may motivate the design of potential therapeutics, deliverable to previously overlooked target sites within aortic smooth muscle, and ultimately novel treatments for aging-induced cardiovascular disease. / 2017-10-27T00:00:00Z

1. Abstract ARP2/3 complex is well studied in case of animals, it plays key roles in motility of cells and intracellular organels. It's malfunctions result in severe growth disorders and even lethality of affected cells. On the contrary, plant cells do not exhibit such dramatic phenotype of ARP2/3 complex mutations like it is by animals. It is possible that just the different life strategies of plants and animals contribute to differences in a way how animal and plant cells use their cytoskeleton, where ARP2/3 complex is it's part as well. It is highly conserved 7 protein complex from yeast to human. His main functions are creation of new "de novo" actin filaments, actin branched filaments network. Some of the parasite organisms are capable of missusing its nucleator activity to actively move inside of host cell. Because of the plant cells are sourounded by the cell wall, which give them support in creating various shapes and also hinders active movement of the whole cell body, it is likely that ARP 2/3 complex could be possibly involved in novel plant specific functions as well. If we think about the different life strategy of plants and animals we can not ignore all the things these two kingdoms have in common regarding to cytoskeleton processes. That is the need both for vesicular transport and...

Metastatic cancer is more dangerous and difficult to treat than pre-metastatic cancer. Ninety percent of cancer-related deaths are caused by metastatic cancer. When cells go through metastases, they go through changes that allow them to break away from the primary tumor and invade secondary tissues. These changes, in lipid membrane composition and cellular glycocalyx, make the cell more resistant to therapeutics. Actin cytoskeleton contractility plays a major role in these changes, as increased contractility has been linked to upregulation of phosphoinositides and production of glycoproteins. Light induced molecular adsorption of proteins (LIMAP) was used to control the actin arrangement and cell shape in order to mimic and study metastatic cells. Negatively charged proteins electrostatically adhere to the surface in order to create patterns for the cells to stick. Neutravidin was conjugated to poly(glutamic acid) to improve attachment to the surface. We observed differences in cell shape and phosphoinositide behavior based on LIMAP patterning. Additionally, expression of key glycoproteins related to cancer metastasis increased with increased actin contractility. The actin cytoskeleton was the main driver of changes to the cell membrane and glycocalyx.

Classically considered as a soluble system of enzymes, glycolysis does not conform to the known function and subcellular microcompartmentation pattern. Certain glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be found at different cellular locations in animal cells, where it exhibited its non-glycolytic activities. Determination of the subcellular localization of two cytosolic GAPDH isoforms from Arabidopsis thaliana (GapC1 and GapC2), fused to Fluorescent Proteins (FP), was investigated in the transiently transformed mesophyll protoplasts, using confocal Laser Scanning Microscopy. Apart from its cytosolic distribution, the nuclear compartmentation of GapC:FP was observed in this study, as well as its punctuate or aggregate-like localization. Part of the GapC:FP foci were observed as mitochondria-associated. A further yeast two-hybrid screen with both GapC isoforms as baits allowed to identify the mitochondrial porin (VDAC3; At5g15090) as a protein-protein interaction partner. Further tests with one-on-one yeast two-hybrid and Bimolecular Fluorescence Complementation (BiFC) assays showed that the detected binding between plant VDAC3 and GapC in yeast cells was false positive. Interestingly, aldolase interacted with VDAC3, as well as with GapC in plant protoplasts, using the BiFC method. On the other hand, no such interaction could be detected in the one-on-one yeast two-hybrid assay. Thus, a model of indirect mitochondrial association of GapC via aldolase that binds directly to mitochondrial porin is proposed to occur only upon certain cellular conditions. Attempts to show the binding of Arabidopsis GAPDH to the actin cytoskeleton in vivo failed, whereas in vitro cosedimentation assays demonstrated that the fully active, recombinant glycolytic enzyme binds to rabbit F-actin. Moreover, is the presence of the GapC cofactor NAD and a reducing agent (DTT), the enzyme might exhibit an actin-bundling activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Knowledge of the molecular and cellular mechanisms of ischemic injury is necessary for understanding acute kidney injury and devising optimal treatment regimens. The cortical actin cytoskeleton in the proximal tubule epithelial cells of the kidney nephron, playing an important role in both the establishment and maintenance of cell polarity, is drastically disrupted by the onset of ischemia. We found that in LLC-PK cells (a porcine kidney proximal tubule epithelial cell line), cortactin, an important regulator of actin assembly and organization, translocated from the cell cortex to the cytoplasmic regions upon ischemia/ATP-depletion. Meanwhile both the tyrosine phosphorylation level of cortactin and cortactin’s interaction with either F-actin or the actin nucleator Arp2/3 complex were down-regulated upon ischemia/ATP-depletion or inhibition of Src kinase activity. These results suggest that tyrosine phosphorylation plays an important role in regulating cortactin’s cellular function and localization in the scenario of kidney ischemia. The Rho GTPase signaling pathways is also a critical mediator of the effects of ATP depletion and ischemia on the actin cytoskeleton, but the mechanism by which ATP depletion leads to altered RhoA and Rac1 activity is unknown. We propose that ischemia and ATP depletion result in activation of AMP-activated protein kinase (AMPK) and that this affects Rho GTPase activity and cytoskeletal organization (possibly via TSC1/2 complex and/or mTOR complex). We found that AMPK was rapidly activated (≤5 minutes) by ATP depletion in S3 epithelial cells derived from the proximal tubule in mouse kidney, and there was a corresponding decrease in RhoA and Rac1 activity. During graded ATP-depletion, we found intermediate levels of AMPK activity at the intermediate ATP levels, and that the activity of RhoA and Rac1 activity correlated inversely with the activity of AMPK. Activation of AMPK using two different drugs suppressed RhoA activity, and also led to morphological changes of stress fibers. In addition, the inhibition of AMPK activation partially rescued the disruption of stress fibers caused by ATP-depletion. This evidence supports our hypothesis that the activation of AMPK is upstream of the signaling pathways that eventually lead to RhoA inactivation and cytoskeletal dysregulation during ATP-depletion.

Development is punctuated by morphogenetic rearrangements of epithelial tissues, including complete detachment of individual motile cells during epithelial-mesenchymal transition (EMT). Dramatic actin rearrangements occur as cell-cell junctions are dismantled and cells become independently motile during EMT. Characterizing dynamic actin rearrangements and identifying actin machinery driving these rearrangements is essential for understanding basic mechanisms of cell-cell junction remodeling; yet, neither the precise series of actin rearrangements at cell-cell junctions that accompany EMT, nor the machinery that controls actin rearrangement during EMT, have been identified. This work represents a detailed study of junctional actin reorganization in cells undergoing EMT, identifies actin regulatory proteins that control this actin reorganization, and defines the specific function of one regulatory protein, zyxin, in EMT. Using immunofluorescence and live cell imaging of HGF induced scattering of MDCK cells, dynamic actin rearrangement events occurring during EMT are characterized. Junctional actin characteristic of cell-cell adherent cells is rearranged into contractile medial actin networks linked to the junctional membrane in the initial steps of cell scattering. This actin rearrangement is accompanied by dynamic redistribution of specific actin regulatory proteins, namely α-actinin and zyxin-VASP complexes. α-Actinin mediates higher order structure of junctional actin. Zyxin-VASP complexes mediate linkage of dynamic medial actin networks to adherens junction membranes. Zyxin regulation of actin-membrane linkage controls whether cell migration during EMT occurs independently in solitary cells or is coordinated through tissues. The functional analysis employed here uses novel, quantitative methods that define specific cellular EMT ‘phenotypes’ to reveal the precise role of zyxin in EMT. Constitutive active zyxin mutants exhibit persistent actin-membrane linkages and a scattering phenotype in which cells migrate without loss of cell-cell adhesion. Zyxin is proposed to regulate EMT progression by regulating disruption or maintenance of actin membrane linkages at cell-cell junctions. Zyxin alters the ability of cells to fully detach and migrate independently during EMT and may be an important regulator of morphogenetic plasticity.

Epithelial morphogenesis is contingent upon cell shape changes. Cell shape changes are the driving force for the metamorphosis of the adult Drosophila leg from the leg imaginal disc precursor. Genetic analysis has identified several Drosophila genes involved in regulating cell shape changes during leg disc morphogenesis. These include members of the RhoA signaling pathway and the product of the Stubble-stubbloid (Sb-sbd) locus, a transmembrane serine protease. Mutations in the Sb-sbd gene interact genetically with the members of the RhoA signaling pathway, however the nature of the relationship between Sb-sbd serine protease activity and RhoA signaling is not understood. To identify additional components of the RhoA signaling pathway that may help us to understand the role of the Sb-sbd protease in RhoA signaling the Drosophila genome was systematically scanned for genes that interact with Sb-sbd and RhoA mutations using deletions/deficiencies of specified regions of each chromosome. A total of 201 deficiencies uncovering approximately 84.9-91% of the euchromatic genome and spanning the X, second, and third chromosoms were tested. Of the 201 deficiencies tested, five putative interacting genetic regions and one gene within these deficiencies were identified. The candidate gene Eip78C encodes a nuclear steroid hormone receptor previously identified as having an important role in metamorphosis.

<p>The actin cytoskeleton is involved in many essential cellular events such as mitosis, cell migration, control of epithelial barrier function, and adherence of immune cells. Given the essential roles of the actin cytoskeleton, it is not surprising that it is a common target for bacterial virulence factors. <em>Legionella pneumophila</em>, the causative agent of Legionnaire’s pneumonia, establishes a replicative compartment using effectors secreted by its Dot/Icm secretion system. At least four Dot/Icm effectors, VipA, Ceg14, LegK2 and RavK have been shown to modulate the host actin cytoskeleton. Here, we identified RavJ (lpg0944) as an additional effector that interferes with the actin cytoskeleton in mammalian cells. We demonstrated that RavJ is a transglutaminase that functions to induce crosslink between actin and members of the Motin protein family, leading to inhibition of the binding between actin and ADF/cofilin. We also found that LegL1 (lpg0945) is a metaeffector of RavJ, which suppresses the transglutaminase activity of RavJ by blocking its enzymatic domain. </p>

Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for human genital and ocular infections. Species of Chlamydia utilize a type-III secretion system to deliver bacterial effector proteins into the host cell in order to promote invasion and establish residence within a parasitophorous vacuole called an inclusion. The effector protein Tarp has been previously implicated as an important effector for promoting invasion during Chlamydia trachomatis infection by directing the formation of new actin filaments and bundles. Intriguingly, the significance of Tarp mediated cytoskeletal changes has not been fully explored in vivo. Host-pathogen interaction studies that replicate the human infection can be performed with mouse adapted Chlamydia, Chlamydia muridarum. However, the genetic tools to create gene deletions in C. muridarum have been lacking. Recently, our collaborators in the Fields and Wolf Laboratories developed a novel genetic tool for creating Tarp deletion mutants and complement clones in Chlamydia muridarum. Through the use of this tool, we were able to study the significance of Tarp in a murine infection model. In addition to Tarp, two other early effectors TmeA and TmeB are hypothesized to play a role in invasion, but a full account of their involvement remained unknown. In our studies, we were able to determine the roles of TmeA and TmeB in remodeling the host cytoskeleton. Using biochemical crosslinking assays, and actin polymerization studies, we discovered that TmeA has the ability to activate host protein N-Wasp in order to increase Arp2/3-dependent actin polymerization, while TmeB can in turn inhibit Arp2/3-directed actin polymerization via direct interactions with Arp2/3. Collectively, these are important findings as our studies have revealed how a collection of early chlamydial effectors work to modulate the host cytoskeleton to facilitate Chlamydia infections.

The Cx43 CT mimetic peptide, αCT1, originally designed to bind to ZO1 and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing(ECIS), a TRITC-dextran transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell-cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell-cell border localization of endothelial VE-cadherin, the Tight Junction protein Zonula Occludens 1 (ZO1) , and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1-binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema. / Doctor of Philosophy / Endothelial cells make up blood vessels within the heart and regulate the exchange of fluids between the circulation and heart tissue. In many forms of heart disease, the cardiac endothelium is disrupted, resulting in a damaging leakage and buildup of fluids within the heart. This work explores how a small peptide, derived from a naturally occurring molecule, may help to prevent fluid-associated damage to the heart by stabilizing the blood endothelium.