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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.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

Small GTPases are important proteins that affect many cellular processes. In my work I compare the five most important protein families of small GTPases - Arf, Rab, Ran, Ras and Rho to identify amino acids responsible for major functional differences between different protein families. To compare them, I have used the structural data from the PDB database and sequences from the UniProt database. I have discovered previously undescribed groups of amino acids specific for each protein family of small GTPases with the help of programs ConSurf and Sca5. I also carried out a pilot study of the applicability of B-factors as indicators of bond strength in the protein structure on the example of small GTPases. The first results are not entirely conclusive, but they do not exclude the applicability of B-factors as indicators of bond strength either. Powered by TCPDF (www.tcpdf.org)

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

c-Cbl functions as a multifunctional adaptor and an E3 ubiquitin protein ligase. Several studies have shown that c-Cbl is involved in cytoskeleton-mediated events, but the molecular mechanisms linking c-Cbl to cytoskeletal rearrangements remain to be elucidated. Our previous results indicated that c-Cbl facilitates spreading and migration of v-Abl-transformed NIH 3T3 fibroblasts and suggested that small GTPases play important roles in the cytoskeletal effects of c-Cbl in this system. To elucidate the individual contributions of small GTPases to these effects, we assessed the roles of endogenous Rac1, RhoA and Rap1 in the c-Cbl-dependent spreading and migration of v-Abl-transformed fibroblasts overexpressing c-Cbl, using RNAi. Furthermore, since it has been shown that Rap1 can act as an upstream regulator of Rac1 in inducing cell spreading, we analyzed the interplay between Rap1 and Rac1 in the signaling pathways connecting c-Cbl to the cytoskeletal events. Our results indicate that Rac1 is essential for cell migration and spreading, whereas activation of RhoA exerts a negative effect. We have also shown that Rap1 is essential for cell spreading, although not for migration in our experimental system. Furthermore, we provide evidence that Rap1 is located upstream of Rac1 in one of the signaling pathways that regulate c-Cbl-facilitated cell spreading. Overall, our findings are consistent with the model describing the connection of c-Cbl to the cytoskeletal rearrangements via two pathways, one of which is mediated by PI3K and Rac1, and the other, by CrkL/C3G, Rap1 and Rac1. A major biological feature of glioma is the ability to invade normal brain tissue. The molecular mechanisms of glioma invasion are involved in multiple biological processes which are primarily associated with cytoskeleton-mediated events including adhesion, migration, degradation of extra cellular matrix (ECM). Biological functions of c-Cbl in glioma have not been elucidated. In this study, we examined biological roles of c-Cbl using RNAi-mediated depletion of endogenous c-Cbl and stably c-Cbl expressing glioma cells generated by lentiviral transduction and showed that c-Cbl increases invasion through degradation of ECM by upregulation of MMP2 but not through migration, adhesion, or growth of SNB19, a grade IV glioblastoma cell line. / Microbiology and Immunology / Accompanied by two .avi videos

The dopamine signalling pathway has been implicated in the pathophysiology of neuropsychiatric conditions including bipolar disorder and schizophrenia. A detailed analysis of this pathway is essential for understanding these conditions. Previous work (Zhan et al., 2008) has identified eleven novel human dopamine receptor interacting proteins (DRIPs), but their role in cell signalling remains unclear. In this project we have employed a biomedical model, Dictyostelium discoideum, to help elucidate the cellular signalling of two DRIPs, the Zizimin GEF (DRIP2) and MARK (DRIP9) proteins. Bioinformatics analysis of these proteins shows conservation of the domain structure in the human and Dictyostelium gene products. To investigate the function of these proteins during development, two Dictyostelium homologues within each family (ZizA and ZizB; MrkA and MrkC) were ablated and changes in developmental for resulting null mutants were analysed. Development was unaltered following ablation of zizA, mrkA and mrkC, however, ablation of zizB gave rise to a clear change in developmental morphology. To further understand the developmental defect of zizB, directional cell movement (chemotaxis) was analysed in the zizA and zizB null mutants. Ablation of zizA caused no gross phenotypic change in chemotaxis, whereas zizB ablation gave rise to a reduction in cell speed, directionality and aspect (roundness). Furthermore, expression studies showed zizA and zizB were constantly expressed throughout development. Overexpression of each gene (labelled with the fluorescent tag, GFP) demonstrated a cytosolic localisation the gene products, with the ZizB-GFP fusion protein additionally exhibiting enrichment of the cortex, causing a large increase in filopodia formation and a partial inhibition of cytokinesis. Analysis of protein binding partners for ZizB indicates specific interaction with Rac1 A and a range of actin-interacting proteins. In conclusion this project provides the first insight into the molecular and cellular functions of Zizimin proteins, potential dopamine receptor interacting protein.

Pseudomonas aeruginosa is an opportunistic pathogen that is a serious problem for immuno-compromised patients. Toxins such as exoenzyme (Exo) S, ExoT, ExoY and ExoU are secreted and translocated from the bacteria into the eukaryotic cell via the bacterial encoded type III secretion system. Our research focuses on ExoS, a bifunctional toxin comprising a Rho-GTPase-activating protein domain (RhoGAP) and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain termed MLD. In this study, cell lines expressing activated forms of various components of the Ras signaling pathway have been used to understand the functional and mechanical activation of ExoS-ADP-ribosyltransferase activity and to reveal its cellular targets in the cell. Our observations suggested that Ras GTPase is the dominant target by which ExoS mediates cell death and activated Ras is able to protect cells against cell death, regardless of whether it has been ADP-ribosylated by ExoS. It has been reported that the 14-3-3 cofactor protein is required for ADP-ribosyltransferase activity of ExoS and a phosphorylation-independent interaction occurs between 14-3-3 and the C-terminal part of ExoS. We have undertaken a deeper analysis including structural and biological investigation of this interaction. Our results suggested that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity. Structural analysis showed that ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. Our structure was supported by biochemical and cytotoxicity analyses, which revealed that the substitution of important residues of ExoS significantly weakens the ability of ExoS to modify endogenous targets such as RAS/RAP1 and to induce cell death. Further, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. Leucine residues-422, 423, 426, and 428 of ExoS are important for the interaction with the ″roof″ of the amphiphatic groove of 14-3-3. In conclusion, we show the mechanism of cell signal transduction pathways affected upon ExoS infection and also demonstrate that the hydrophobic residues of ExoS in 14-3-3 interaction motif have a significant role for ExoS enzymatic activity.

Yes / Efficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif. / Cancer Research UK grants A15672, A24450, and multidisciplinary grant A20017.

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