Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2008. / "A dissertation in molecular biology and biochemistry and cell biology and biophysics." Advisor: Leonard L. Dobens. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Sept. 12, 2008. Includes bibliographical references (leaves 120-147). Online version of the print edition.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Neuroscience; migration; yeast two-hybrid; MIG-10. Includes bibliographical references (leaves 58-60).

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves 97-129. Also available online.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: axon outgrowth; signal transduction; C. elegans; cell migration; mig-10. Includes bibliographical references (p. 61).

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 195 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Low Intensity Pulsed Ultrasound (LIPUS) is used clinically to promote wound healing. In vivo studies show that LIPUS is effective in a wide range of tissue types, and in vitro experiments show that multiple cell types respond to LIPUS stimulation. Despite this, there is no unifying mechanism of how LIPUS stimulation is sensed by cells, and it is unknown what the early signalling events are. The LIPUS signal is a mechanical one; therefore I hypothesised that mechanosensitive organelles, called focal adhesions, would be essential for the induction of cellular signalling events in response to this type of stimulation. Proteins within these structures (such as vinculin and talin) link the actin cytoskeleton to the extracellular matrix via integrins, and are known to be sensitive to mechanical forces, capable of generating intracellular signalling events in response to mechanical stimulation. The purpose of this work was to identify the early signalling events occurring within minutes of LIPUS stimulation; determine the molecular mechanisms behind such events; and to investigate whether such events require integrin-mediated adhesions. In the first part of the work, I established the use of live-cell imaging together with LIPUS stimulation to directly observe the cellular response. I determined rapid reorganizations of the actin cytoskeleton, which led to increased cell velocity. These effects were found to be Rac dependent, and, using FRET-based probes, I measured rapid increases in Rac activity occurring within minutes of LIPUS stimulation. The second part of this work identified an increase in the number of early endosomes in cells stimulated with LIPUS. This phenotype was also Rac dependent, as well as requiring the early endosomal regulator protein Rab5. In this chapter, I observed an increase in the association between Rac and Rab5 in response to LIPUS stimulation, and this contributes to Rac activation. Using substrates to block integrin-mediated adhesion, I determined that cell-matrix adhesions are required for the effects of LIPUS stimulation. Using vinculin-deficient cells, I determined that this mechanosensitive protein is vital for co-ordinating Rac activation in response to LIPUS. In particular, the actin binding tail is needed for mechanosensing of this LIPUS signal. In the final chapter I established the use of photoactivatable (PA) GFP to assess adhesion protein turnover. This technique was used to show that LIPUS stimulation directly affects the turnover of vinculin. Overall, this work shows that the mechanosensitive protein vinculin is crucial for sensing the mechanical stimulation provided by LIPUS, orchestrating downstream Rab5-mediated Rac activation to enhance cell motility.

Migrating cells have two basic ways of extending their leading edge: by dendritic actin polymerization beneath the membrane, or by fluid pressure, which produces blebs. Most cells are believed to move using actin-driven projections, but in more physiological conditions, blebbing motility is also apparent. It has been shown that certain cells even can switch between these two modes of motility, although it is not known how this switch is triggered. Besides, it is unclear whether blebbing can be regulated by chemotactic stimuli, and generally, how blebbing is controlled in the cell. In this study I employed a popular model organism – Dictyostelium discoideum – to investigate the role of blebbing in chemotaxis. Here I confirm that in standard conditions Dictyostelium cells move by a combination of F-actin-driven protrusions and blebs. Blebbing is characterized by the rapid projection of hemispherical patches of plasma membrane at 2-4 times the speed of an actin-driven projection, and leaves transient scars of F-actin marking the original cortex in the base of blebs. I demonstrate that Dictyostelium cells can adjust their mode of movement according to the conditions: in a resistive environment they switch almost entirely to “bleb mode”. I show that in chemotaxing cells, blebs are mainly restricted to the leading edge, and they often lead the way when a cell is forced to re-orientate. Bleb location appears to be controlled directly by chemotactic gradients. To investigate how chemoattractant induces blebbing, I have screened signal transduction mutants for altered blebbing. I have found that blebbing is unaffected in many chemotactic mutants, but unexpectedly depends on PI3-kinases and two downstream PIP3-binding proteins of unknown function – PhdA and CRAC. I conclude that Dictyostelium cells move using a hybrid motor in which hydrostatic pressure-driven bleb formation is as important as F-actin-driven membrane extension, and that cells can change the balance between modes as required. I propose that blebbing motility of Dictyostelium cells is a direct response to mechanical resistance of environment. More generally, bleb-driven motility may be a ‘”high-force” mode of movement that is suited to penetrating tissues. Blebs are chemotactic and their induction may involve branches of the chemotactic signal transduction pathway distinct from F-actin regulation.

No description available.

Cell migration is an essential physiological process required for embryogenesis, tissue repair and immune surveillance. Directional cell migration requires coordinated regulation of multiple integrin-mediated cellular processes, including dynamic regulation of the actin cytoskeleton, precise control of membrane protrusion events and the constant recycling of adhesion receptors. While it is clear that regulation of the small guanosine triphosphatases (GTPases) Rac1, Arf6 and RhoA is critical for these processes, the integrin-dependent mechanisms responsible for cyclic activation and dynamic coordination of GTPase signalling are only partially understood. Here, analysis of three published mass spectrometry (MS) studies cataloguing integrin-dependent adhesion complexes identified filamin-A and IQGAP1 as potential candidates linking β1 integrin to the regulation of Rac1 activity. Using immunoprecipitation, MS analysis, immunocytochemistry and RNAi, filamin-A and IQGAP1 were found to be recruited to integrin activation sites, where they constrained Rac1 activity via the recruitment of the GTPase-activating protein RacGAP1. The functional relevance of Rac1 deactivation, through a RacGAP1 and IQGAP1-mediated mechanism, is to permit efficient membrane protrusion and directional cell migration. Subsequently, IQGAP1 was identified as a molecule co-ordinating Rac1 and Arf6 activities downstream of β1 integrin engagement, via the recruitment of the GTPase activity modulators RacGAP1, srGAP2 and HERC1. This lead us to propose a model whereby IQGAP1, through the recruitment of multiple small GTPase activity modulators, co-ordinates the two small GTPases Rac1 and Arf6, to efficiently regulate directional cell migration. Dyregulated cell migration due to integrin over-activation is associated with tumour invasion. Increased recycling of α5β1 integrin, resulting from expression of mutant p53 or inhibition of αVβ3 integrin function, leads to random cell migration on 2D substrates and promotes tumour invasion via activation of the pro-invasive kinase Akt. Here, the RacGAP1- IQGAP1 complex was identified as a key component of this pathway. In particular, RacGAP1 was found to be phosphorylated by Akt2 on T249, a phosphorylation event that promoted RacGAP1 recruitment to IQGAP1, at the cell front, and triggered cell invasion by inducing a Rac1/RhoA activity switch. These findings demonstrated that Akt activation, downstream of α5β1 integrin recycling, promotes fibronectin-mediated cell invasion by activating a novel RacGAP1/IQGAP1/Rac1/RhoA pathway. Taken together, we identified a novel signalling nexus, downstream of integrin activation and/or recycling that co-ordinate the small GTPases Rac1, Arf6 and RhoA during cell migration and invasion.