Abstract Filamins are large dimeric proteins that cross-link actin into three-dimensional bundles or orthogonal networks. In addition to an actin-binding domain, each filamin monomer contains 24 immunoglobulin-like domains separated by flexible regions between domains 15–16 and 23–24. Dimerisation of filamin occurs through the Ig-like domain 24. Filamins bind to a variety of molecules. They provide a link between the plasma membrane and the cytoskeleton through interactions with transmembrane receptors, and at the same time, serve as a platform for signalling molecules. Filamins are involved in several human diseases affecting the central nervous system, vascular system and muscle. In this study the structure of the the carboxy-terminus of filamin was resolved and details of filamins interaction with a platelet surface protein important in haemostasis were analysed. An x-ray structure of the Ig-like domain 24 of human filamin C was solved at the resolution of 1.43 Å. The asymmetric unit of the crystal contains one monomer; a crystallographic dimer is formed by 2-fold axis symmetry. Point mutation studies confirmed that the dimer seen in the crystal is also present in solution. The structure showed that the dimerisation mode of human filamin is completely different from that in the Dictyostelium discoideum amoeba filamin analogue. Human filamin dimerises through β-strands C and D, and the Dictyostelium protein through β-strands B and G located on the opposite edge of the β-sandwich. Based on the sequence homology between vertebrate filamins it was proposed that the interface seen in human filamin is common for all vertebrate filamins. The structure of human filamin C Ig-like domains 23–24 was solved by combining the techniques of x-ray crystallography and small angle x-ray scattering (SAXS). This structure provides further insight into the organization of the domains in the carboxy-terminal part of filamin molecule. One of the first structural examples of the interaction of filamin with a ligand was provided by this study. The x-ray structure of filamin A domain 17 in complex with the alpha subunit of the GPIb-V-IX receptor was solved at a resolution of 2.3 Å. The interaction between filamin and the GPIbα-V-IX receptor is important for maintaining the integrity and shape of blood platelets, as well as for regulating the receptor adhesive function. This study also revealed that the Ig-like domain 17 represents a major binding site of filamin to GPIbα. The Kd of the interaction, determined by calorimetric studies, was 11 μM. The specificity of the filamin A 17 - GPIbα interaction is mainly determined by hydrophobic contacts.

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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

This work identified an ortholog of Abp1 (actin binding protein 1) in Dictyostelium (Dabp1). In order to analyze the functions of Dabp1 in Dictyostelium, loss-of–function studies and gain-of-function studies were performed by generating cells that either deleted the Dabp1 gene from the genome or overexpressed the Dabp1 protein. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild type cells had a single leading pseudopodium and a single uropod, whereas more than 27% of Dabp1 null cells projected multiple pseudopodia. Similarly, ~ 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium. This work also revealed an interplay between Dabp1 and MyoB, one of the Myosin I proteins, in controlling pseudopodia formation in Dictyostelium. These two proteins colocalize partially at the cortex in growing cells. The peripheral localization of MyoB was dependent on Dabp1. Depletion of both Dabp1 and MyoB caused defects in organization of the actin cytoskeleton and actin related activities such as formation of small F-actin filled spikes on the cell cortex of growing cells, a higher percentage of multinucleated cells, and an increased number of pseudopodia branching extensively. When MyoB was overexpressed in Dabp1 null mutants, cells had similar phenotypes as Dabp1/MyoB double null mutants, and displayed an increased number of pseudopodia with many branches. Overexpression of Dabp1 in MyoB null mutants rescued the defects in pseudopodia formation. The SH3 of Dabp1 was shown to be important for the rescue of defects caused by depletion of MyoB. Collectively, these data suggest that MyoB and Dabp1 work cooperatively to regulate the uniformity and integrity of the actin extensions during chemotaxis. MyoB requires Dabp1 to function in this process. Dabp1 may function as a scaffold to recruit MyoB to the proper localization. These studies of Dabp1 in Dictyostelium raise broad question about functions of actinassociated proteins in pseudopodia formation and the importance of uniformity and integrity for actin structures in chemotaxis. / text

Cancer metastasis accounts for approximately 90% of all cancer-related deaths; however, the underlying mechanisms remain largely unknown. It has been known proteins that control F-actin dynamics are crucial for cancer metastasis. In this study, we revealed how an F-actin binding protein, Switch-associated protein 70 (SWAP-70), contributes to breast cancer metastasis. Moreover, immunotherapy is a promising approach to treat metastatic cancer cells by enhancing the function of the host immune system against cancer. Our lab has conducted extensive studies on how SWAP-70 regulates the function of several immune cell types, including dendritic cells (DCs), B cells, and mast cells. These cells have been reported to contribute to tumor immunity. Thus, we hypothesized that SWAP-70 plays a role in tumor immunity. To characterize the function of SWAP-70 in metastasis, we generated 4T1, mouse breast cancer, SWAP-70 knockout (KO) cells using Crispr/Cas9 technology. A syngeneic orthotopic model was used to recapitulate clinical disease progression, and the results showed that SWAP-70 led to significant metastasis to the lungs and bones in immunocompetent mice. Several functional assays have revealed that SWAP-70 promotes anchorage-independent growth, cell migration, invasion, and adhesion in 4T1 cells. Biophysical measurements showed that SWAP-70 contributes to cellular mechanics. To investigate how SWAP-70 in host cells affects tumor immunity, SWAP-70 deficient mice were injected with E0771 mouse breast cancer cells to study tumorigenicity. SWAP-70 deficient mice showed delayed primary tumor growth and less distant metastasis. Isolated SWAP-70−/− DCs were impaired in generating CD8 T cell responses pulsed with soluble OVA protein, but not with OVA peptide, suggesting that the antigen uptake, processing, and presentation process in SWAP-70−/− DCs may be diminished. Taken together, our findings describe the potential mechanisms by which the loss of SWAP-70 hinders cancer metastasis and provide several insights into how targeting SWAP-70 could be a potential therapeutic approach to target cancer.

Plasmodien spp. sind obligat intrazellulär lebende Parasiten, welche einen evolutionär konservierten aktinabhängigen molekularen Motor für die Fortbewegung und den Wirtszellein- und -austritt nutzen. In dieser Arbeit werden die Aktinregulatoren Adenylyl- Zyklase- assoziierte Protein (C-CAP), Profilin sowie die Aktin depolymerizierenden Faktoren 1 und 2 (ADF1, ADF2) in Plasmodium berghei charakterisiert. Die Geninaktivierung von C-CAP besitzt keinen Einfluss auf die Entwicklung von pathogenen Blutstadien. C-cap(-) Ookineten bewegen sich jedoch deutlich langsamer, sind aber in der Lage den invertebraten Wirt zu infizieren. Defekte treten während der extrazellulären Replikationsphase im Mosquito auf und führen zu Abbruch des Lebenszykluses. Die erfolgreiche Komplementierung der Defekte mit dem orthologen Gen aus Cryptosporidium parvum CpC-CAP bestätigt die funktionale Redundanz zwischen beiden Proteinen. Profilin, als ein weiteres G-Aktin bindendes Protein, ist hingegen nicht in der Lage die Defekte des c-cap(-) Parasiten auszugleichen. Mittels transgener Parasiten welche ein C-CAPmCherry Fusionsprotein exprimieren, wird das C-CAP Protein im Zytoplasma lokalisiert. Erstmals wird mit dieser Arbeit ein G-Aktin bindendes Protein, C-CAP beschrieben, welches eine essentielle Funktion während der Oozystenreifung in Plasmodium berghei besitzt. Die Transkription der Aktinregulatoren Profilin, ADF1 und ADF2 wird in Sporozoiten drastisch herunterreguliert und Profilin kann als Protein nicht mehr nachgewiesen werden. Um die Funktion von C-CAP und Profilin zu überprüfen, wurden beide Proteine spezifisch in Sporozoiten überexprimiert. Diese Parasiten sind nicht in der Lage die Speicheldrüsen des Wirtes zu besiedeln, was zum Abbruch des Lebenszykluses führt. Anhand dieser Ergebnisse entwickele ich ein „minimalistisches“ Model zur Beschreibung der Aktinregulation in Sporozoiten in welchem das ADF1 als regulatorisches Protein im Mittelpunkt steht. / Plasmodium spp. are obligate intracellular parasites, which employ an conserved actin-dependent molecular motor machinery that facilitates their motility, host cell invasion and egress. In this work I report implications of the actin-regulators adenylyl cyclase-associated protein (C-CAP), profilin and actin depolymerization factor 1 and 2 (ADF1, ADF2) in distinct and previously unanticipated cellular processes during the life cycle of in the rodent malarial parasite Plasmodium berghei. Fluorescent tagging of the endogenous C-CAP genetic locus with mCherry revealed cytosolic distribution of the protein. Gene deletion demonstrates that the G-actin binding protein C-CAP is entirely dispensable for the pathogenic blood stages. Ookinetes show reduced motility, but are competent infecting the mosquito host. Defects emerging in the extracellular replication phase, leading to attenuation of oocyst maturation. Successful trans-species complementation with the C. parvum C-CAP ortholog, rescues the c-cap(-) phenotype and proves functional redundancy. The actin regulator profilin fails to rescue the defects of c-cap(-) parasites, despite sharing its actin sequestering activity with C-CAP. Taken together, C-CAP is the first G-actin sequestering protein of Plasmodium species that is not required for motility but performs essential functions during oocyst maturation. Characterization of the actin regulators profilin, ADF1 and ADF2 revealed dramatic transcriptional down-regulation and the absence of the profilin protein in sporozoites. To test whether G-actin binding proteins interfere with sporozoite functions, I ectopically overexpressed of profilin and C-CAP stage-specifically in sporozoites. This conducted to abolishment of salivary gland invasion and lifecycle arrest. Based on these unexpected findings and the available literature data, I developed a “minimalistic model” for actin regulation in sporozoites that predicts ADF1 as the main actin-turnover regulating factor.