Actin filaments and microtubules are involved in cell development and morphogenesis. Plant Class II formins regulate both cytoskeletal polymers. However their function has not yet been fully described. This study examines effects of LOF mutations in Arabidopsis thaliana FH13 (AT5G58160) and FH14 (AT1G31810) genes on early root system development using a pharmacological approach. Since measuring root length of numerous mutant lines in multiple conditions is laborious and time consuming, this thesis also involves optimization of this process with the aim to establish a reliable method of fast visualisation and measurement of Arabidopsis seedlings in a time series in the laboratory. Furthermore, statistical analysis for a large amount of data gathered in multiple conditions had to be optimized. While no significant phenotype in terms of root length was found in fh13, fh14 and double fh13 fh14 LOF mutants under standard conditions, treatment with cytoskeletal drugs revealed possible changes in lateral root branching in an fh14 mutant. Nevertheless, specific function of FH13 and FH14 remains a question.

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Formin proteins, important regulators of a cell's actin cytoskeleton, nucleate actin polymerization and promote filament elongation. Actin dynamics are crucial for a form of polarized growth termed tip growth that is performed by cells involved in reproduction and nutrient uptake in plants. Uncovering the molecular basis of how actin associated proteins like formins control actin dynamics is important to gain a fundamental understanding of plant growth mechanisms. In the moss Physcomitrella patens, there are 9 formin genes that group into three distinct classes (I, II and III). From previous work, we suspect that class I formins may play a role in cytokinesis. Thus, I investigated how class I formins localize in tip-growing protonemal cells to gain further insight into their function. To do this, I tagged class I formins with GFP at the endogenous locus and visualized their subcellular localization using confocal microscopy. I found that Formin 1A, 1D, 1E and 1F localize to punctate spots on the plasma membrane and may concentrate at the cell plate during cell division, while 1B localizes to the cytosol. Overall, these data have shown that class I formins may play a role in cell division and potentially in the secretory pathway.

Indiana University-Purdue University Indianapolis (IUPUI) / Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, has been implicated in the non-canonical Wnt mediated Planar Cell Polarity (PCP) signaling pathway. Although the studies in Drosophila Daam1 and Xenopus Daam1 generated inconsistent conclusions regarding the function of Daam1, the biological function of mammalian Daam1 was not evaluated. In this study, we used a mouse promoter trap technology to create Daam1 deficient mice to analyze the role of Daam1 in embryonic development and organogenesis. Daam1 is highly expressed in the developing heart. The majority of Daam1 mutant mice died between embryonic day 14.5 and birth, exhibiting a variety of heart defects, which include ventricular noncompaction, ventricular septal defects, and double outlet right ventricle. About 10% mutant mice survive to adulthood, and these survivors do not show significantly compromised heart function based on echocardiographic analyses. However, all of these mutant survivors have ventricular noncompaction with a range of severities. A conditional rescue experiment using a cardiac specific Cre mouse line, Nkx2-5Cre, confirmed that the cardiac defects are the primary cause of death in Daam1 mutants. Both in vivo and ex vivo analyses revealed that Daam1 is essential for regulating non-sarcomeric filamentous actin assembly in cardiomyocytes, which likely contributes to cardiac morphogenesis and ventricular wall maturation. Biochemical studies further suggested that Daam1 is not a key signaling component in regulating the activation of small GTPases, such as RhoA, Rac1 and Cdc42. In conclusion, our studies demonstrated that Daam1 is essential for cardiac morphogenesis likely through its regulation of cytoskeletal architecture in the developing cardiomyocytes. / indefinitely

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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.

Dynamic actin remodeling is at the core of a number of fundamental cellular processes in a variety of organisms ranging from animal neuronal outgrowth to pollen tube growth during plant reproduction and asymmetrical cell division in budding yeast. Such dynamism results from a concerted effort of a number of temporally and spatially regulated actin binding proteins. The polymerization and depolymerization of elaborate F-actin networks takes place by the addition and removal of actin subunits at the two filament ends with each end possessing distinct properties, making the filaments themselves polar structures with a fast growing “barbed end” and a slow growing “pointed end”. Although actin polymerization at the barbed end is an efficient and favorable process, the initial filament nucleation step is a much more inefficient and thermodynamically unfavorable process requiring the need for a variety of actin nucleating proteins. These nucleating proteins not only determine the precise location of actin assembly at the cell membrane during polarized cell growth but also directly control the number of force producing filaments. Here we have concentrated our efforts in characterizing a number of Arabidopsis thaliana , Group I membrane bound, formin homologues AFH1, AFH5, AFH6, AFH7, AFH8 and AFH11 using a range of functional genomics tools. Consequently we have shown that AFH5 is a pollen expressed actin nucleating protein localized at the tip of the polarized tube. Genetic alterations of the AFH5 gene using T-DNA gene knockout and gene overexpression both show distinct deformities at the tip of the rapidly growing pollen tube leading to inefficient fertilization during plant reproduction and reduced silique length. In the tip growing root hair cells of Arabidopsis the afh5 mutants show a wavy and bulgy phenotype at the hypocotyl region while the afh1 mutants project branched, split root hairs along the primary root. Although our results do indicate that AFH1 and AFH5 are expressed in the polarized pollen tubes and root hairs their, expression and hence activity is spatially controlled and restricted to different parts of the cell.

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