Chemotaxis is a process whereby single- and multi-cellular organisms migrate in response to external chemical stimuli. This directed cell movement is regulated by complex signaling pathways and is implicated in embryonic development, immune response, and the metastasis of cancer cells. Dictyostelium discoideum, social amoebae with the ability to migrate and aggregate in response to chemoattractants such as cAMP, have been used as a model system to study chemotaxis. Preliminary research suggests that protein kinase (PKA) is involved in some of the signaling pathways that regulate chemotaxis. The role of PKA in chemotaxis was investigated, first, by characterizing the phenotype of PKA null cells using established cell biological and biochemical assays. Furthermore, spatiotemporal regulation of critical cytoskeletal proteins was probed in wild-type and PKA null cells using confocal fluorescence microscopy, indicating misregulation of both F-actin and Myosin II in pkaC- and pkaR- cells. Finally, preliminary work was done to lay the groundwork for experiments exploring possible PKA targets mediating TORC2 function in chemotaxis.

The actin cytoskeleton is critical to the proper function of cells and its misregulation can lead to human disease states. As a consequence, actin dynamics is tightly controlled. To gain further insight into the mechanisms controlling actin dynamics, my studies have focused on two families of proteins implicated in actin regulation. The Nck proteins act as molecular adaptors in signal propagation by linking upstream mediators, which they recognize through the Nck SH2 domain, to downstream effectors, which bind the Nck SH3 domains. I have found that Nck is required in podocyte cells for proper foot process formation, a process involving actin cytoskeletal reorganization, and therefore for proper kidney function. Furthermore, I show that Nck links the podocyte adhesion protein nephrin to actin polymerization. In cell-based assays, nephrin-induced actin polymerization is dependent on an interaction with functional Nck, which occurs through binding of three phosphorylated tyrosine sites within the cytoplasmic tail of nephrin to the Nck SH2 domain. Finally, I demonstrate that the enteropathogenic E.coli protein Tir reorganizes the cytoskeleton by molecular-mimicry of nephrin-like signaling. The srGAP proteins are GTPase activating proteins that attenuate the activity Rho GTPases, proteins directly involved in actin cytoskeletal control. The regulatory mechanisms that control srGAP activity are unclear. I have found that the srGAP family members srGAP1, srGAP2, and srGAP3 interact, through their carboxy-terminal region with 14-3-3 proteins, and that this interaction is dependent on protein kinase C-induced phosphorylation of srGAP. 14-3-3 binding does not affect the activity of srGAP2, as determined using cell-based GAP assays. Further studies are required to clarify the biological significance of this interaction to srGAP regulation. The data presented in this thesis furthers our understanding of signaling networks that control the actin cytoskeleton, and brings us closer to the goal of fully understanding actin dynamics and cellular signaling.

The actin cytoskeleton is critical to the proper function of cells and its misregulation can lead to human disease states. As a consequence, actin dynamics is tightly controlled. To gain further insight into the mechanisms controlling actin dynamics, my studies have focused on two families of proteins implicated in actin regulation. The Nck proteins act as molecular adaptors in signal propagation by linking upstream mediators, which they recognize through the Nck SH2 domain, to downstream effectors, which bind the Nck SH3 domains. I have found that Nck is required in podocyte cells for proper foot process formation, a process involving actin cytoskeletal reorganization, and therefore for proper kidney function. Furthermore, I show that Nck links the podocyte adhesion protein nephrin to actin polymerization. In cell-based assays, nephrin-induced actin polymerization is dependent on an interaction with functional Nck, which occurs through binding of three phosphorylated tyrosine sites within the cytoplasmic tail of nephrin to the Nck SH2 domain. Finally, I demonstrate that the enteropathogenic E.coli protein Tir reorganizes the cytoskeleton by molecular-mimicry of nephrin-like signaling. The srGAP proteins are GTPase activating proteins that attenuate the activity Rho GTPases, proteins directly involved in actin cytoskeletal control. The regulatory mechanisms that control srGAP activity are unclear. I have found that the srGAP family members srGAP1, srGAP2, and srGAP3 interact, through their carboxy-terminal region with 14-3-3 proteins, and that this interaction is dependent on protein kinase C-induced phosphorylation of srGAP. 14-3-3 binding does not affect the activity of srGAP2, as determined using cell-based GAP assays. Further studies are required to clarify the biological significance of this interaction to srGAP regulation. The data presented in this thesis furthers our understanding of signaling networks that control the actin cytoskeleton, and brings us closer to the goal of fully understanding actin dynamics and cellular signaling.

Cofilin’s activity to nucleate actin filament assembly, is regulated by phosphorylation at a single site on the amino terminus, Serine 3. Phosphorylation at this site abolishes the ability of ADF/cofilin to bind to F-actin and inhibits its severing function. This work characterizes the ability of dephosphorylated cofilin (mutation at Serine 3 site) to navigate prostate cancer actin cytoskeleton and metastatic properties in response to TGF-β. TGF-β increased Lim Domain Kinase 2 (LIMK-2) activity leading to cofilin phosphorylation and decrease actin filament severing in wild type cofilin (WTCFL) PC-3 cells. Constitutively active cofilin in Serine 3 cofilin mutants (S3ACFL) promoted prostate cancer cell filopodia formation, actin severing and directed TGF-β mediated migration and invasion. Co-culture of prostate cancer cells with prostate cancer associated fibroblasts induced cell invasion in WTCFL and S3ACFL cells. Active cofilin further enhanced the invasive response, even in the presence of a TGF-β-neutralizing antibody, implicating the contribution of the microenvironment. Active cofilin led to a significant increase in prostate cancer cell metastatic potential in vivo and cofilin correlated with metastasis in a mouse model of prostate tumor progression. In human prostate cancer, cofilin expression was significantly higher in metastasis compared to the primary tumors. Cofilin thus emerges as a regulator of the actin cytoskeleton remodeling capable of coordinating the cellular responses to TGF- β towards prostate cancer metastasis. Understanding how cancer cells interprete TGF-β signals from the microenvironment, is critical for defining the mechanism via which TGF- β function is switched from a growth suppressor to a metastasis promoter. Here we show that in prostate cancer, TGF-β action is directed by active cofilin enabling actin cytoskeleton changes and metastatic behavior. The significant association of cofilin with prostate cancer metastatic progression supports its predictive and targeting value in metastasis.

<p>Essential and diverse biological processes such as cell division, morphogenesis and migration are regulated by a family of molecular switches called Rho GTPases. These proteins cycle between active, GTP-bound states and inactive, GDP-bound state and this cycle is regulated by families of proteins called Rho GEFs and GAPs. GAPs are proteins that stimulate the intrinsic GTPase activity of Rho-family proteins, potentiating the active to inactive transition. GAPs target specific spatiotemporal pools of GTPases by responding to cellular cues and utilizing protein-protein interactions. By dissecting these interactions and pathways, we can infer and then decipher the biological functions of these GAPs.</p><p>This work focuses on the characterization of a novel Rho-family GAP called srGAP2. In this study, we identify that srGAP2 is a Rac-specific GAP that binds a Formin-family member, Formin-like 1 (FMNL1). FMNL1 is activated by Rac and polymerizes, bundles and severs actin filaments. srGAP2 specifically inhibits the actin severing of active FMNL1, and the assembly of an srGAP2-FMNL1 complex is regulated by Rac. Work on FMNL1 shows that it plays important roles in regulating phagocytosis and adhesion in macrophages. To learn more about srGAP2 and its role in regulating FMNL1, we studied macrophages isolated from an srGAP2 KO mouse we have recently generated. This has proven quite fruitful: loss of srGAP2 decreases the ability for macrophages to invade through extracellular matrix but increases phagocytosis. These results suggest that these two processes might be coordinated in vivo by srGAP2 and that srGAP2 might be a critical regulator of the innate immune system.</p> / Dissertation

Internal and external signaling dependent changes in cell behavior are directly linked to force-generating remodeling of the actin microfilament system which is juxtaposed to the inside of the plasma membrane. This dynamic filament system is involved in many processes in the cytoplasm and the nucleus of eukaryotic cells.   This thesis studies profilin, a regulator of actin filament dynamics which functions during incorporation of new actin molecules at growing filament ends at the cell periphery. Profilin is also present in the nucleus but its function is less well understood in this compartment. Here I present results concerning profilin and the activity of the transcription factor SRF, which is known to control the expression of actin and many actin-binding proteins in a process requiring the MRTF-A co-factor. MRTF-A binds monomeric actin and is released upon receptor mediated actin polymerization. Depletion of the two profilin isoforms I and IIa reduced MRTF-A/SRF-dependent transcription, most likely since the lack of profilin enable more MRTF-A to bind actin monomers and thereby prevent SRF-transcription. Interestingly profilin depletion also seemed to affect general transcription in the two cell lines investigated. In a separate study, a close connection between profilin, and possibly also profilin:actin, with microtubules was revealed. Microtubules are important for intracellular trafficking of vesicles as well as directional cell migration and the observation made here suggests the existence of a microtubule-associated platform for actin filaments formation. In congruence, the microtubule-associated actin nucleation promoting factor WHAMM was found to interact with profilin. Finally, the intracellular distribution of profilin was investigated by fluorescence microscopy using different peptide specific antibodies. Since these antibodies showed unique but varying results our work emphasizes common problems connected with this technique. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper1: Manuscript; Paper 2: Manuscript; Paper 3: Manuscript</p>

Elements that are foreign to the human body, such as bacteria, viruses and fungi, are recognised by cells of the innate immune system. Through a process termed phagocytosis, microorganisms are bound, internalised and destroyed. In this thesis, we focus upon how host cells respond to IgG-opsonised targets, studying both the initial stages of Fc-receptor (FcR) ligation and the later stages of phagocytic cup formation. We provide evidence that after clustering of the receptors, the mobility of diacylated probes such as those found in Src-family of kinases, was reduced. This immobilisation was found to be insensitive to cholesterol depletion, arguing against a role for conventional ‘lipid rafts’ in the initiation of receptor signalling. Furthermore, decreased mobility was only partially dependent upon the presence of actin which could provide a physical restriction. Importantly, inhibiting Src-family kinase activity, completely abrogated immobilisation. These results are highly suggestive of a previously unrecognised mechanism for the initation of FcR signalling. At later stages, receptor-derived signalling leads to the formation of an actin-rich phagocytic ‘cup’. We found that even before a large particle was fully internalised, actin cleared from the base of the phagocytic cup. This clearance was necessary for the internalisation of large particles, as chemically stabilising actin prior to clearance, abrogated internalisation. Actin clearance was shown to be the indirect result of the localised disappearance of phosphatidylinositol 4,5-bisphosphate and the dephosphorylation of tyrosine-phosphorylated proteins. Strikingly, phosphatidylinositol 3-kinase activity was required for both the protein dephosphorylation and for the phosphatidylinositol 4,5-bisphosphate hydrolysis that was responsible for actin disassembly. We propose that actin disassembly is required to recycle actin to the advancing pseudopods, in order to complete phagocytosis. For many microorganisms, internalisation through phagocytosis means certain death. Obligate intracellular bacteria, such as Salmonella enterica serovar Typhimurium however, can readily survive inside host cells. This is achieved through modulation of the host-cell signalling pathways that normally lead to microbial destruction. In S. Typhimurium, a needle-like complex, delivers small protein effectors that aid in the survival of the bacterium. We studied one such effector: SigD, that had been suggested to have phosphatidylinositol phosphatase activity. Indeed, we showed that when the cDNA for SigD was exogenously expressed in mammalian cells, phosphatidylinositol 4,5-bisphosphate was depleted and phosphatidylinositol 5-phosphate was formed. We characterised the physiological effects of this 4-phosphatase activity and furthermore, describe the use of SigD as a research tool for modulating host cell phospholipids.

A central process in connective tissue homeostasis is cell migration, which involves dynamic interactions between focal adhesions, the actin cytoskeleton and mitochondria, but the role of focal adhesion proteins in cell migration is not wholly defined. We examined focal adhesion-associated proteins from mouse fibroblasts and identified Flightless I (FliI) as a potential focal adhesion protein. We determined that FliI is distributed in the cytosol and co-localizes with actin monomers and mitochondria, but partially with paxillin. Biochemical assays showed that FliI associates with both actin monomers and short oligomers/filaments. Migration assay determined that cells with reduced FliI expression migrated more quickly and that FliI knockdown inhibited activation of β1 integrins. Consistent with these data, cell adhesion assay demonstrated that FliI knockdown cells were less adherent than wildtype cells. Our findings indicate that FliI may regulate cell migration by interacting with the actin monomers and the mitochondria to affect cell adhesion.

Elements that are foreign to the human body, such as bacteria, viruses and fungi, are recognised by cells of the innate immune system. Through a process termed phagocytosis, microorganisms are bound, internalised and destroyed. In this thesis, we focus upon how host cells respond to IgG-opsonised targets, studying both the initial stages of Fc-receptor (FcR) ligation and the later stages of phagocytic cup formation. We provide evidence that after clustering of the receptors, the mobility of diacylated probes such as those found in Src-family of kinases, was reduced. This immobilisation was found to be insensitive to cholesterol depletion, arguing against a role for conventional ‘lipid rafts’ in the initiation of receptor signalling. Furthermore, decreased mobility was only partially dependent upon the presence of actin which could provide a physical restriction. Importantly, inhibiting Src-family kinase activity, completely abrogated immobilisation. These results are highly suggestive of a previously unrecognised mechanism for the initation of FcR signalling. At later stages, receptor-derived signalling leads to the formation of an actin-rich phagocytic ‘cup’. We found that even before a large particle was fully internalised, actin cleared from the base of the phagocytic cup. This clearance was necessary for the internalisation of large particles, as chemically stabilising actin prior to clearance, abrogated internalisation. Actin clearance was shown to be the indirect result of the localised disappearance of phosphatidylinositol 4,5-bisphosphate and the dephosphorylation of tyrosine-phosphorylated proteins. Strikingly, phosphatidylinositol 3-kinase activity was required for both the protein dephosphorylation and for the phosphatidylinositol 4,5-bisphosphate hydrolysis that was responsible for actin disassembly. We propose that actin disassembly is required to recycle actin to the advancing pseudopods, in order to complete phagocytosis. For many microorganisms, internalisation through phagocytosis means certain death. Obligate intracellular bacteria, such as Salmonella enterica serovar Typhimurium however, can readily survive inside host cells. This is achieved through modulation of the host-cell signalling pathways that normally lead to microbial destruction. In S. Typhimurium, a needle-like complex, delivers small protein effectors that aid in the survival of the bacterium. We studied one such effector: SigD, that had been suggested to have phosphatidylinositol phosphatase activity. Indeed, we showed that when the cDNA for SigD was exogenously expressed in mammalian cells, phosphatidylinositol 4,5-bisphosphate was depleted and phosphatidylinositol 5-phosphate was formed. We characterised the physiological effects of this 4-phosphatase activity and furthermore, describe the use of SigD as a research tool for modulating host cell phospholipids.

A central process in connective tissue homeostasis is cell migration, which involves dynamic interactions between focal adhesions, the actin cytoskeleton and mitochondria, but the role of focal adhesion proteins in cell migration is not wholly defined. We examined focal adhesion-associated proteins from mouse fibroblasts and identified Flightless I (FliI) as a potential focal adhesion protein. We determined that FliI is distributed in the cytosol and co-localizes with actin monomers and mitochondria, but partially with paxillin. Biochemical assays showed that FliI associates with both actin monomers and short oligomers/filaments. Migration assay determined that cells with reduced FliI expression migrated more quickly and that FliI knockdown inhibited activation of β1 integrins. Consistent with these data, cell adhesion assay demonstrated that FliI knockdown cells were less adherent than wildtype cells. Our findings indicate that FliI may regulate cell migration by interacting with the actin monomers and the mitochondria to affect cell adhesion.