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.

During inflammation, leukocytes modulate α4β1(VLA-4) integrin avidity in order to rapidly stabilize nascent adhesive contacts to VCAM-1-expressing endothelial cells and resist detachment forces imparted by the flowing blood. Linkage to the actin cytoskeleton is critical for integrin function, yet the exact role of the actin cytoskeleton in leukocyte adhesion stabilization under conditions of fluid flow remains poorly understood. We modeled leukocyte (U937 cell, mouse lymphocyte and human monocyte) arrest and adhesion stabilization through the use of a parallel plate flow chamber and visualized cells by phase contrast or fluorescent confocal microscopy. Live cell imaging with Lifeact-transfected U937 cells revealed that mechanical forces imparted by fluid flow induced formation of upstream tension-bearing anchors attached to the VCAM-1-coated surface. Scanning electron microscopy confirmed that flow-induced mechanical force culminates in the formation of structures that anchor monocyte adhesion. These structures are critical for adhesion stabilization, since disruption of actin polymerization dramatically inhibited VLA-4-dependent resistance to detachment, but did not affect VLA-4 expression, affinity modulation, and clustering or constitutive linkage to F-actin. Transfection of dominant-negative constructs and inhibition of kinase function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. Rap1 was shown to be critical for resistance to flow-induced detachment and accumulated in its GTP form at the sites of anchor formation. A key mediator of force-induced Rac activation and actin polymerization is PI3K. Live cell imaging revealed accumulation of PIP3 within tension-bearing anchors and blockade of PI3K or deficiency of PI3Kγ isoform reproduced the adhesion defect produced by inhibition of actin polymerization. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces; these included activation of Rap1, phosphoinositide 3-kinase γ-isoform and Rac, but not Cdc42.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

The object of this thesis is to present two novel applications of Spatiotemporal Image Correlation Spectroscopy (STICS) to biological systems. STICS is a technique which uses the correlations in pixel intensity fluctuations of an image time series, captured under fluorescence microscopy, to measure the speed and direction of a flowing population of fluorescently labeled molecules. The method was first applied to measure the dynamics of transport vesicles inside growing pollen tubes of lily flowers. The measured vector maps allowed to confirm the presence of actin filaments along the periphery of the tubes, as well as the presence of a reverse-fountain pattern in the apical region. In a second set of experiments, STICS was used to measure the retrograde flow of filamentous actin in migrating chick DRG neuronal growth cones. These results serve as proof of principle that STICS can be used to probe the response of the growth cone cytoskeleton to external chemical cues.