Formine der Cappuccino-Familie nukleieren lineare Aktinfilamente mittels der formin homolgy 2- (FH2-) Domäne, Spir-Proteine mittels des WASP-Homologie-Domäne 2- (WH2-) Clusters. spire- und cappuccino-Mutanten haben in Drosophila einen nahezu identischen Phänotyp. Zudem wurde ein überlappendes Expressionsmuster der Säugerhomologe von Drosophila spire- und cappuccino, spir-1 und formin 2, im sich entwickelnden und im adulten Zentralnervensystems von Mäusen beobachtet. In dieser Arbeit wurde eine mögliche Interaktion von Spir-Proteinen mit Forminen der Cappuccino-Familie aus Drosophila und Maus in in vitro Bindungsstudien und in in vivo Kolokalisationsstudien untersucht. Eine direkte Interaktion wurde für Drosophila Spir und Cappuccino sowie für Säuger Spir-1 und Formin-2 nachgewiesen. Die Interaktionsdomänen sind konserviert und wurden auf die Kinase Non-catalytic C-lobe Domain (KIND-Domäne) der Spir-Proteine und auf die FH2-Domäne der Cappuccino-Proteine eingegrenzt. Diese Interaktion ist spezifisch für Spir- und Cappuccino-Proteine. So interagiert die FH2-Domäne des Formins Diaphanous-1 nicht mit Spir-1-KIND. Ebenso interagiert die KIND-Domäne des RasGEFs very-KIND (VKIND) nicht mit der FH2-Domäne von Formin-2. / Formins of the Cappuccino-family and Spir proteins are nucleators of unbranched actin filaments. Cappuccino proteins mediate nucleation by means of the formin homology 2 (FH2) domain, Spir proteins via a cluster of four WASP homology domains 2 (WH2). The Drosophila spire- and cappuccino- mutant phenotypes are nearly identical. Furthermore, the mammalian orthologs of Drosophila spire- and cappuccino, spir-1 and formin-2 have an overlapping expression pattern in the developing and adult nervous system of mice. The work presented here focused on a potential protein interaction of Cappuccino-subfamily formins and Spir proteins which was analyzed using in vitro binding assays and in vivo colocalization studies. A direct interaction between Drosophila Spir and Cappuccino as well as between mammalian Spir-1 and Formin-2 was determined. The binding sites were narrowed down to the Kinase Non-catalytic C-lobe Domain (KIND) domain of Spir and the FH2 domain of Cappuccino-subfamily formins. This conserved interaction is specific for Spir- and Cappuccino proteins. Both, the in vivo and the in vitro studies did not reveal any interaction of Spir-1-KIND with the FH2-domain of the formin Diaphanous-1 nor for Formin-2-FH2 with the KIND domain of the RasGEF very-KIND (VKIND).

Cancer cell metastasis is induced by actin-dependent cell migration and is affected by cytoskeletal remodelling proteins. FMNL2 is one such protein which promotes colorectal cancer (CRC) cell metastasis and amoeboid style invasion of melanoma cells. FMNL2 mRNA is subject to alternative splicing and studies suggest that the resulting encoded proteins are likely to differ in their regulation, subcellular localization and activity. We identified four FMNL2 isoforms (ITM, YHY, PMR and TQS) expressed in non-invasive (SW480) and invasive (SW620) CRC cells, as well as in highly invasive A375 amoeboid melanoma cells. qPCR data suggests that an “invasive” isoform (TQS) may be preferentially expressed in highly invasive and amoeboid cell lines. Boyden chamber invasion assay results show that FMNL2 knockdown inhibits amoeboid style invasion in two melanoma cell lines and that TQS is the most efficient isoform at rescuing the invasive phenotype. This study provides a further understanding of FMNL2’s role in invasion and metastasis and identifies specific targets for the development of future antimetastatic therapies.

The actin and microtubule cytoskeleton play critical roles in Golgi and cilia assembly. Inverted-Formin 1 (INF1) is a novel, microtubule-associated protein that regulates both actin and microtubule dynamics and affects Golgi and cilia assembly. A non-biased discovery based approach was used to investigate the interactome of INF1 using BioID in combination with stable isotopic labeling in cell culture (SILAC). A number of INF1-interacting proteins were identified and validated in co-IP experiments. The INF1 interaction domains were mapped using an extensive set of INF1 deletion and point mutation derivatives. Functional characterization of these interactions suggests a mechanism for the effects of INF1 on ciliogenesis. The establishment and maintenance of cellular architecture requires the coordinated, dynamic regulation of actin and microtubule networks. Our data suggests that INF1 plays a crucial role in connecting these two cytoskeletal networks for the regulated assembly of the Golgi ribbon and the primary cilium.

Cancer cell metastasis is induced by actin-dependent cell migration and is affected by cytoskeletal remodelling proteins. FMNL2 is one such protein which promotes colorectal cancer (CRC) cell metastasis and amoeboid style invasion of melanoma cells. FMNL2 mRNA is subject to alternative splicing and studies suggest that the resulting encoded proteins are likely to differ in their regulation, subcellular localization and activity. We identified four FMNL2 isoforms (ITM, YHY, PMR and TQS) expressed in non-invasive (SW480) and invasive (SW620) CRC cells, as well as in highly invasive A375 amoeboid melanoma cells. qPCR data suggests that an “invasive” isoform (TQS) may be preferentially expressed in highly invasive and amoeboid cell lines. Boyden chamber invasion assay results show that FMNL2 knockdown inhibits amoeboid style invasion in two melanoma cell lines and that TQS is the most efficient isoform at rescuing the invasive phenotype. This study provides a further understanding of FMNL2’s role in invasion and metastasis and identifies specific targets for the development of future antimetastatic therapies.

A fundamental yet unresolved issue in cell biology is how force regulates actin dynamics and how this biophysical regulation is modulated by biochemical signaling molecules. Here we show, by atomic force microscopy (AFM) force-clamp experiments, that tensile force regulates the kinetics of G-actin/G-actin and G-actin/F-actin interactions by decelerating dissociation at low forces (catch bonds) and accelerating dissociation at high forces (slip bonds). The catch bonds can be structurally explained by force-induced formation of new interactions between actin subunits (Steered molecular dynamics (SMD) simulations performed by Dr. Jizhong Lou, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China). K113S mutation on yeast actin suppressed the actin catch-slip bonds, supporting the structural mechanism proposed by SMD simulations. Moreover, formin controlled by a RhoA-mediated auto-inhibitory module can serve as a "molecular switch", converting the catch-slip bonds to slip-only. These results imply anisotropic stability of the actin network in cells subjected to directional forces, possibly explaining force-induced cell and actin fiber alignment controlled by RhoA and formin. Our study suggests a molecular level crosstalk mechanism bridging the actin-mediated mechanotransduction and biochemical signal transduction pathways.

No description available.

Establishment of polarity is a critical process that occurs early during development. In Drosophila melanogaster, axis determination occurs by localization of determinants during oogenesis. Mutations in cappuccino (capu) lead to defects in polarity establishment of both the anterior/posterior (A/P) and dorsal/ventral (D/V) axes during oogenesis. In the oocytes laid by capu mutant females, determinants that define these axes are either mislocalized or are absent. Several lines of evidence suggest that the regulation of cytoskeleton by the gene product encoded by capu is involved in Drosophila oogenesis.Capu, a member of the formin family of proteins, known to be regulators of actin dynamics, interacts both genetically and physically with chickadee (chic) which encodes the actin binding protein Profilin. I show here that mutations in both capu and chic lead to defects in the endocytic uptake of yolk into developing oocytes. I show that mutations in these loci lead to accumulation of abnormally large yolk granules and that this is a post internalization defect in the oocyte of capu and chic females. I also present evidence which indicates that an interaction with capu is necessary for chic regulation of yolk granule biogenesis.This is the first evidence for a formin subfamily of formin proteins to have a role in endocytosis. While this new function identified for the actin associated proteins Capu and Profilin indicates that regulation of actin cytoskeleton plays a role in endocytosis during oogenesis, the mechanism of this regulation and possible actin independent roles played by Capu and Profilin in this process are yet to be determined.Like capu, mutations in spire (spir) also show defects in A/P and D/V axes during oogenesis. Spir is an actin binding protein and like capu, mutations in spir shows defects in cytoskeletal architecture and suggests that capu and spire alter microtubule distribution in the oocyte during oogenesis.To identify molecular partners of capu and spir and their roles during oogenesis, I performed a genome-wide deficiency screen to identify regions of the genome that interact with these genes. I identified regions in the genome that showed interaction with capu and spir. While I was able to narrow down the region of interaction to a smaller cytological interval, gaps in the deficiency coverage and lack of mutants in those regions prevented me from identifying interacting loci in those regions.

Formins are potent regulators of actin dynamics that can remodel the actin cytoskeleton to control cell shape, cell cytokinesis, and cell morphogenesis. The defining feature of formins is the formin homology 2 (FH2) domain (Paul and Pollard, 2008), which promotes actin filament assembly while processively moving along the polymerizing filament barbed end. INFT-1 is one of six formin family members present in Caenorhabditis elegans (Hunt-Newbury et al., 2007) and is most closely related to vertebrate INF2, an inverted formin with regulatory domains in the C- rather than N-terminus. Nematode INFT-1 contains both formin homology 1 (FH1) and formin homology 2 (FH2) domains. However, it does not share the regulatory N-terminal Diaphanous Inhibitory Domain (DID) domain and C-terminal Diaphanous Autoregulatory Domain (DAD) domain found in mammalian INF2. In contrast to mammalian INF2, the sequence of INFT-1 starts immediately at FH1 domain and C-terminal region of INFT-1 shares little homology with INF2, suggesting that elegans INFT-1 is regulated by other mechanisms. We used fluorescence spectroscopy to determine the effect of INFT-1 FH1FH2 on actin assembly and total internal reflection fluorescence microscopy to investigate how INFT-1 formin homology 1 and formin homology 2 domains (FH1FH2) mediate filament nucleation and elongation. INFT-1 FH1FH2 nucleates actin filament and promote actin assembly. However, INFT-1 FH1FH2 reduces filament barbed-end elongation rates in the absence or presence of profilin. Evidences demonstrated that INFT-1 is non-processive, indicating a unique mechanism of nucleation. INFT-1 nucleation efficiency is similar to the efficiency of Arabidopsis FORMIN1 (AFH1), another non-processive formin. High phosphate affected the assembly activity of INFT-1 FH1FH2 in the absence or presence of profilin. INFT is thus the second example of a non-processive formin member and will allow a more detailed understanding of the mechanistic difference between processive and non-processive formins. / Master of Science

This thesis investigates whether there are alternative mechanisms of tip growth in invasive and non-invasive hyphae of the fungus Neurospora crassa. The cytoskeleton protein actin is thought to play a pivotal role in hyphal tip growth, performing a multitude of tasks, one of which may be the provision of a resistive force to counter turgor pressure. An Actin depleted zone (ADZ) was the dominant feature of invasive hyphal tips, which was largely absent from non-invasive hyphae. The Spitzenkörper was slightly larger in invasive hyphae but this size difference alone was thought insufficient to account for the exclusion of filamentous actin (F-actin) from the tip. The actin nucleating protein formin was found at sites where actin nucleation is occurring, while cofilin, a protein that severs F-actin, was found to localise where F-actin disassembly was likely to be occurring. It is suggested that these proteins are likely to play a role in controlling a dynamic cytoskeleton, rearrangements of which are required for the two modes of growth. Invasive hyphae were found to generate a higher turgor than non-invasive hyphae. These results suggest that the F-actin rearrangements facilitated by cofilin give an ADZ that may play a role in invasive hyphal tip growth; possibly through a reduction of tip resistance; thus enabling the provision of a greater protrusive force by turgor.

<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