Universiẗat, Diss., 2005--Osnabrück.

Universiẗat, Diss., 2004--München.

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.

WASH complex regulates actin dynamics on endosomes by activating the Arp2/3 complex, which subsequently induces generation of branched actin patches. WASH complex is required for proper recycling of many important transmembrane proteins. Although the general physiological function of WASH complex is known, the role of its single subunits have not yet been adequately specified. This work focuses on one of these subunits - protein SWIP. This protein maintains vesicular localization of some WASH complex subunits in the slime mold Dictyostelium discoideum and a point mutation in its sequence causes a severe neurodegenerative disease - autosomal recessive intellectual disorder (ARID). Our results show that SWIP truncation results in its inability to incorporate into WASH complex. However, the C-terminal part of SWIP is able to localize onto intracellular vesicles, which are not WASH complex positive. We have also studied the impact of ARID-causing SWIP mutation, and we show, that it does neither change the protein's ability to bind the complex nor the overall localization of WASH complex.

Epithelia act as protective barriers and it is therefore essential that wounded epithelia are rapidly repaired to maintain barrier function. Cells surrounding epithelial wounds become motile following wounding, which involves generating dynamic actin structures that drive closure of the wound. These actin structures include filopodia which are important in the final stage of epithelial closure in which the opposing epithelial edges are joined together. The molecular mechanisms that trigger wound edge cells to become motile are not well understood. Using Drosophila wound healing and the morphogenetic process dorsal closure as models, we find that phosphatidylinositol 3,4,5-triphosphate (PIP3) regulates epithelial closure by promoting the formation of filopodia at epithelial edges. PIP3 accumulates at epithelial edges and genetically depleting PIP3 results in reduced filopodia and defects in epithelial closure. We demonstrate that the GTPase Rac and guanine nucleotide exchange factor Myoblast City function downstream of PIP3 to promote filopodia formation. We also demonstrated that the scaffolding protein Par3/Bazooka and the lipid phosphatase PTEN are responsible for restricting the localisation of PIP3 and consequently the downstream signals to the epithelial leading edge, so acting to determine the location of filopodia formation. This project reveals a novel mechanism by which actin protrusions, required for epithelial closure, are formed in response to epithelial damage. Additionally, we have identified an additional role for PIP3 in regulating the extrusion of cells from epithelial sheets in the Drosophila embryo. This finding implicates PIP3 in the regulation of tissue homoeostasis, and could contribute to our understanding of tumour initiation as unregulated tissue growth can result in the formation of tumours.

It is becoming clear that mechanical stimuli are critical in regulating cell biology; however, the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells expressing actin-EGFP with controlled forces (0-20nN) in order to investigate the cell’s structural response. Two clear force dependent responses were observed: a short-term local deformation of actin stress fibres and a long-term force-induced remodelling of stress fibres at cell edges, far from the point of contact. We were also able to quantify strain dynamics occurring along stress fibres. The cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres, indicating localized dynamic contraction and expansion. A ~50% increase in myosin contractile activity is apparent following application of 20nN force. Directly visualizing force-propagation and stress fibre strain dynamics has revealed new information about the pathways involved in mechanotransduction which ultimately govern the downstream response of a cell.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22464号 / 工博第4725号 / 新制||工||1738(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 梅田 眞郷, 教授 水落 憲和, 准教授 菅瀬 謙治 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Some myelodysplastic syndrome (MDS) patients present with macrothrombocytopenia due to impaired megakaryocyte (MK) differentiation. Transgenic mice that express the NUP98-HOXD13 (NHD13) fusion gene is a model for MDS and recapitulates the key features of MDS. The study investigated the hypothesis that expression of NHD13 disrupts actin architecture during MK differentiation leading to macrothrombocytopenia. To test the hypothesis, sternums were stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 bone marrow (BM) contained many dysplastic MK. BM from wild type (WT) and NHD13 mice were also flushed, cultured in media supplemented with thrombopoietin only or with estrogen to induce proplatelet formation, and MK harvested after 5 days. Harvested MK and BM cores were processed and analyzed by transmission electron microscopy (TEM) to detail the ultrastructural features. TEM of MK revealed that NHD13 leads to formation of an irregular demarcation membrane system and fewer proplatelets. Cultured WT and NHD13 MK were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin, α/β-tubulin and myosin IIa, and their cytoskeleton compared. Interestingly WT MK had actin either distributed evenly or predominantly in the periphery of the cytoplasm, NHD13 MK displayed only the former phenotype. Additionally, proplatelets lacked actin cytoplasmic extensions. The results from the present thesis demonstrate actin expression and architecture are impaired in dysplastic MK expressing the NHD13 leukemic fusion gene and leads to macrothromcytopenia. Understanding the molecular mechanisms of abnormal MK differentiation in MDS is important as many MDS patients die of hemorrhagic complications. / Master of Science

The actin fibrils found at the ectoplasm-endoplasm interface in Nitella internodal cells are a major component of the mechanism that is responsible for cytoplasmic streaming in these giant algal cells. The fibrils have been shown to attach to the inner surface of internodal chloroplasts which are embedded in long files within the stationary ectoplasm along the length of the cell. The existence of actin bundles at the ends of the cell, where chloroplast files are absent, has not been examined. Through the use of scanning and transmission electron microscopy, the present work shows that actin bundles are continuous throughout the cell and that those bundles in the chloroplast-free endwall region have a distinct distribution from those associated with chloroplast files. Additionally, the organization of regenerated actin bundles in blue light-irradiated areas of cells {in which an area of the cell cortex is stripped of its chloroplasts and associated actin fibrils) is compared to untreated regions of the cell. These morphological observations are quantified and discussed in terms of their implications towards the nature of actin bundle immobilization and bundle organization during cell ontogeny.

Indiana University-Purdue University Indianapolis (IUPUI) / Insulin resistance, a hallmark of type 2 diabetes (T2D), is associated with accompanying derangements such as hyperinsulinemia that promote the progression of insulin resistance, yet a mechanism(s) is imperfectly understood. Data have demonstrated that hyperinsulinemia promotes insulin resistance as evidenced by diminished ability of insulin to mobilize glucose transporter GLUT4 to the plasma membrane (PM). We found that loss of PM phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) structure contributes to hyperinsulinemia-induced insulin resistance. We tested if increased glucose flux through hexosamine biosynthesis pathway (HBP) causes dysregulation of PM components necessary for GLUT4 translocation. Increased HBP activity was detected in 3T3-L1 adipocytes cultured in hyperinsulinemia (5 nM Ins; 12 h) and also 2 mM glucosamine (GlcN), a distal HBP activator, inducing losses of PM PIP2 and F-actin. In accordance with HBP flux directly weakening PIP2/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored F-actin and insulin responsiveness. Furthermore, less invasive challenges with glucose led to PIP2/F-actin dysregulation. New findings support a negative correlation between PM cholesterol accrual, PIP2/F-actin structure and GLUT4 regulation. These data stemmed from parallel study aimed at understanding the antidiabetic mechanism of the nutrient chromium (Cr3+). We found that chromium picolinate (CrPic) enhanced insulin-stimulated GLUT4 trafficking via reduction in PM cholesterol. In line with glucose/cholesterol toxicity findings, we demonstrated that therapeutic effects of CrPic occurred solely in adipocytes with increased HBP activity and a concomitant elevation in PM cholesterol. Mechanistically, data are consistent with a role of AMP-activated protein kinase (AMPK) in CrPic action. These data show that CrPic increases AMPK activity and perhaps suppresses cholesterol synthesis via distal phosphorylation and inactivation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), a rate-limiting enzyme in cholesterol synthesis. Continued study of the consequence of increased HBP activity revealed alterations in cholesterogenic transcription factors – Sp1, SREBP-1, and NFY – with Sp1 showing a significant increase in O-linked glycosylation. Consistent with Sp1 modification eliciting maximal transcriptional activation of SREBP-1, Hmgr mRNA was significantly enhanced. In conclusion, these data are consistent with a central role of PM cholesterol in glucose transport and suggest perturbations in this lipid have a contributory role in developing insulin resistance.