Timely and spatially controlled endosomal trafficking and signaling is important for cell proliferation, directed cell migration, and cell invasion, which are frequently misregulated in cancer cells. Cdc42-interacting protein-4 (CIP4) family adaptors promote endocytosis by inducing membrane invaginations via their Fer/CIP4 Homology-Bin/Amphyphysin/Rvs (F-BAR) domains, coupled with activation of the actin assembly machinery to promote vesicle fusion or motility. My thesis focuses on defining the roles of CIP4, and a related protein, Transducer of Cdc42-mediated actin assembly-1 (Toca-1), in regulating Epidermal Growth Factor Receptor (EGFR) endocytosis, EGFR trafficking, cancer cell motility, and invasion. In Chapter 2, I show that CIP4 and Toca-1 localize to early endosomes and promote EGFR trafficking from early endosomes to lysosomes for degradation, thus limiting extracellular signal-regulated kinase signaling from early endosomes and proliferation of A431 carcinoma cells. In Chapter 3, I provide novel evidence that depletion of Toca-1 results in defects in actin-based lamellipodial protrusions that are required for cell motility. The cause of these defects may relate to altered recruitment of the Abelson-interactor-1 and its effector Wiskott-Aldrich syndrome protein family verprolin-homologous protein to the lamellipodia in A431 cells depleted of Toca-1. Results in Chapter 4 identify CIP4 as a negative regulator of breast cancer invasiveness downstream of Src protein-tyrosine kinase. Src is a potent inducer of extracellular matrix (ECM)-degrading structures called invadopodia that function in tissue invasion by cancer cells. I found that CIP4 is a Src substrate that localizes to Src-induced invadopodia in MDA-MB-231 breast cancer cells. Interestingly, depletion of CIP4 results in enhanced ECM degradation, invadopodia formation, and invasiveness compared to control cells. Thus, CIP4 and Toca-1 are multifaceted regulators of EGFR downregulation, EGF-induced cell motility, and Src-induced cell invasion. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-08-25 11:44:46.934

FCH/CIP4 homology-Bin-Amphiphysin-Rvs (F-BAR) domain proteins are a subfamily of the Bin-Amphiphysin-Rvs (BAR) superfamily of proteins. They contain unique domains that bind and reshape the phospholipid bilayers of endosomal compartments during endocytosis. Using a functional assay for cell survival, we identified an F-BAR protein, FCH/CIP4 homology and double Src homology 3 domains 2 (FCHSD2), that confers drug resistance. Stable expression of shRNA against FCHSD2 in multiple cell types showed that loss of FCHSD2 sensitized cells to apoptosis by doxorubicin. Silencing of FCHSD2 also enhanced the ability of fibroblasts to grow colonies in culture. Mass spectrometry analysis of FCHSD2 protein complexes identified multiple interacting proteins that are involved in adhesion and endosome trafficking. We identified and confirmed a novel interaction between FCHSD2 and sorting nexin 18 (SNX18), a BAR domain protein that binds to endosomes. Our results suggest that FCHSD2 is involved in regulating cellular adhesion and cell death.

FCH/CIP4 homology-Bin-Amphiphysin-Rvs (F-BAR) domain proteins are a subfamily of the Bin-Amphiphysin-Rvs (BAR) superfamily of proteins. They contain unique domains that bind and reshape the phospholipid bilayers of endosomal compartments during endocytosis. Using a functional assay for cell survival, we identified an F-BAR protein, FCH/CIP4 homology and double Src homology 3 domains 2 (FCHSD2), that confers drug resistance. Stable expression of shRNA against FCHSD2 in multiple cell types showed that loss of FCHSD2 sensitized cells to apoptosis by doxorubicin. Silencing of FCHSD2 also enhanced the ability of fibroblasts to grow colonies in culture. Mass spectrometry analysis of FCHSD2 protein complexes identified multiple interacting proteins that are involved in adhesion and endosome trafficking. We identified and confirmed a novel interaction between FCHSD2 and sorting nexin 18 (SNX18), a BAR domain protein that binds to endosomes. Our results suggest that FCHSD2 is involved in regulating cellular adhesion and cell death.

Chez l’ensemble des organismes, la capacité des cellules à se polariser est une propriété nécessaire à la croissance, à la division, à la mobilité, à la différenciation ou encore au trafic intracellulaire. Les GTPases de la famille Rho jouent des rôles prépondérants dans la régulation de ces mécanismes. Chez la levure Saccharomyces cerevisiae, nous étudions la manière dont le régulateur RhoGAP Rgd1p des protéines Rho3 et Rho4 est lui-même régulé au cours de la croissance polarisée. Nous avons montré que la protéine Rgd1 est localisée au fond du bourgeon lors de la croissance isotropique du bourgeon puis sous forme d’un anneau au cou du bourgeon lors de la cytocinèse. Nous avons également montré que le trafic intracellulaire, notamment l’exocytose et les phosphoinosotides tels que le PdtIns(4)P et le PdtIns(4,5)P2 ont un rôle majeur dans la régulation spatio-temporelle de la protéine Rgd1. Ces mécanismes permettraient d’acheminer la protéine RhoGAP au niveau des sites de croissances afin d’agir sur les protéines Rho3 et Rho4. / In all organisms, the ability of cells to polarize is a property necessary for growth, cell division, mobility, cell differentiation or intracellular trafficking. GTPases of the Rho family play central roles in the regulation of these mechanisms. In the yeast Saccharomyces cerevisiae, we study how the regulator RhoGAP Rgd1p of Rho3 and Rho4 proteins may be itself regulated during polarized growth. We showed that the protein Rgd1 is located at the bud tip during isotropic growth of the bud and form a ring at the bud neck during cytokinesis. We also showed that the intracellular traffic, especially the exocytosis and phosphoinosotides as the PdtIns(4)P and PdtIns(4,5)P2 have a major role in the spatio-temporal regulation of the Rgd1 protein. These mechanisms would deliver the RhoGAP protein at growth sites to act on Rho3 and Rho4 proteins.

京都大学 / 0048 / 新制・論文博士 / 博士(理学) / 乙第12911号 / 論理博第1547号 / 新制||理||1590(附属図書館) / 32121 / 京都大学大学院理学研究科・物理学・宇宙物理学専攻 / (主査)講師 市川 正敏, 教授 山本 潤, 教授 田中 耕一郎 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DGAM

Regulation of the actin cytoskeleton is key to cellular function and underlies processes including cell migration, mitosis and endocytosis. Motile cells send out dynamic actin protrusions that enable them to sense and interact with their environment, as well as generating physical forces. Linking of the actin cytoskeleton to the cell membrane is essential for the formation of these protrusions. The proteins that are thought to fulfil such a role have a membrane interacting domain (such as the PH domain in lamellipodin, or I-BAR protein in IRSp53) and a domain which interacts with actin regulatory proteins (such as the SH3 domain of IRSp53, which binds Ena and VASP). I investigated the contribution of the F-BAR protein Toca-1 in linking actin polymerisation to membranes, by characterising a new protein-protein interaction and the interaction of Toca-1 with giant unilamellar vesicles. FBP17, a homologue of Toca-1, can oligomerise to form 2D flat lattices and 3D tubules on membranes. Proteins of the Toca-1 family have previously been implicated in actin polymerisation in cell-free systems and during endocytosis. However, there is emerging evidence that Toca-1 family proteins could also be involved in the formation of outward facing protrusions, lamellipodia and filopodia. In an in vitro system that recapitulates the formation of filopodia-like structures (FLS) on supported lipid bilayers, Toca-1 is recruited early, suggesting a Toca-1 scaffolding mechanism could precede the recruitment of other actin regulators. One prediction of this model is that Toca-1 would bind proteins previously implicated in filopodia formation, such as formins. I found that extracts depleted of Toca-1 binding partners no longer forms filopodia-like structures and subsequently optimised pull-down assays to identify Toca-1 binding partners by mass-spectrometry. I identified four formins, Diaph1, Diaph3, FHOD1 and INF2, and as well as the actin elongation factors and filopodia proteins, Ena and VASP. I further characterised these interactions and found that Toca-1 binds Ena and VASP via its SH3 domain. The interaction is direct and is strongly reduced if the proline-rich region in Ena is deleted. VASP was still able to bind without its proline rich region, suggesting there could be additional binding sites. I discovered that the binding of Ena and VASP was dependent on the clustering state of Toca-1, whilst the binding of the previously identified Toca-1 binding partner N-WASP was not. This further supports the importance of Toca-1 oligomerisation in actin polymerisation. I tested these interactions in the FLS system and found that increasing Toca-1 concentration leads to increased recruitment of N-WASP, as well as the novel binding partner Ena to the structures, whereas an increase in VASP was not observed. SH3-domain mediated interactions are required for Toca-1 recruitment to FLS, suggesting that its membrane and protein binding activities act cooperatively. I showed that unlike N-WASP, which promotes the formation of branched actin, Ena and VASP are not required for actin polymerisation on supported lipid bilayers, suggesting that they are redundant with other factors in the elongation step of FLS formation. Ena and VASP are known to be important for the formation of neuronal filopodia and so I began to further test the role of these interactions in a cellular context using a neuronal cell culture system. As well as recruiting protein binding partners, F-BAR family proteins are implicated in stabilising lipid microdomains and can induce the clustering of phosphoinositides. I investigated the role of Toca-1 in actin polymerisation on PI(4,5)P2-rich giant unilamellar vesicles (GUVs). Actin-rich tails formed on the GUVs only when excess Toca-1 was supplemented into the extracts, and I propose that this is due to lipid organisation by Toca-1. In summary, my work suggests a model in which Toca-1 clusters, stabilises the membrane lipids and recruits regulators of actin polymerisation, such as Ena. This mechanism could be used to link actin polymerisation to the membrane in cellular protrusions, such as filopodia.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24983号 / 生博第512号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 荒木 崇, 教授 鈴木 淳, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM