Thesis (Ph. D.)--University of Oregon, 2004. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 68-73). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--Lehigh University, 1996. / Includes vita. Includes bibliographical references (leaves 78-90).

Thesis (doctoral)--Umeå Universitet, 1985. / Added t.p. with thesis statement inserted. Bibliography: p. 35-41.

Thesis (M.S.)--Brigham Young University. Dept. of Exercise Sciences, 2006. / Includes bibliographical references.

Thesis (M. Sc.)--University of Canterbury, 2004. / Typescript (photocopy). "June 2004." Includes bibliographical references (leaves 105-113). Also available via the World Wide Web.

The tubulin/FtsZ superfamily contains a large set of proteins that spans through all kingdoms of life, with αβ-tubulins being the eukaryotic representatives and FtsZ being the best studied prokaryotic homologue. It is believed that all tubulin/FtsZ-related proteins have evolved from a common ancestor, however, members from this superfamily have diverged in many aspects. αβ-tubulins polymerise into giant and hollow microtubules in the presence of GTP. Despite the size of around 25 nm wide, microtubules display sophisticated dynamics. In particular, dynamic instability, the stochastic change between fast growth and rapid shrinkage, is a hallmark of microtubules. In contrast to αβ-tubulins, FtsZ lacks the C-terminal domain of tubulins and it probably functions as single homopolymeric protofilaments, possibly through treadmilling dynamics. There is strong divergence of the biological functions in the tubulin/FtsZ superfamily. Microtubules are involved in fundamental processes such as motility, transport and chromosomal segregation, whereas FtsZ is involved in bacterial cytokinesis (bacterial cell division), and the equivalent role of FtsZ is carried out by actin-based and ESCRTIII-based systems in eukaryotes. It seems that there is a big evolutionary gap between αβ-tubulins and FtsZ, and the only properties that are conserved within the tubulin/FtsZ superfamily are fold, protofilament formation and GTPase activity. In 2002, a pair of tubulin-like genes, btuba and btubb were identified in Prosthecobacter bacteria, with higher sequence homology to eukaryotic tubulins than FtsZ or any other bacterial homologues. The crystal structures solved later revealed, again, a closer similarity to αβ-tubulins than to their prokaryotic equivalents. It has been known for a while that BtubAB form filaments in the presence of GTP, however, little knowledge has been available regarding the filament architecture. In this project, I determined the near atomic resolution structure of the in vitro BtubAB filament using cryoEM and cryoET, revealing a hollow tube that consists of four protofilaments. A closer look showed that BtubAB filaments have many conserved microtubule features including: an overall polarity, similar longitudinal contacts, M-loops in lateral interfaces, and the presence of the seam, a structural hallmark of microtubules. My study also shows that BtubC, a protein with a TPR fold, binds to the BtubAB filaments in a stoichiometric manner, similar to some MAPs on microtubules. Based on this work, I concluded that BtubAB from Prosthecobacter form bacterial ‘mini microtubules’, and my work provided interesting insight into the evolution of tubulin/FtsZ-related proteins.

Abstract The function of the vertebrate nervous system is based on the rapid and accurate transmission of electrical impulses. The myelin sheath is a lipid-rich membrane that envelops the axon, preventing the leakage of the nervous impulse to the environment. Myelin is formed when the plasma membrane of a myelinating glial cell differentiates and wraps around an axon. The compaction of myelin leads to the extrusion of most of the glial cell cytoplasm from the structure. Both the compact and noncompact regions of myelin carry distinct subsets of proteins. The myelin-associated glycoprotein (MAG) is present in noncompact myelin. It is a cell adhesion molecule expressed only by myelinating glial cells. Two isoforms of MAG, S- and L-MAG, exist, and these forms differ from each other only by their cytoplasmic domains. Until now, little information has been available on the differences between the MAG isoforms. This study was carried out in order to gain information on the cytoplasmic domains of S- and L-MAG. Significant differences were observed in the properties of the MAG cytoplasmic domains. An interaction between the L-MAG cytoplasmic domain and the S100b protein was characterised, and a role for this interaction was found in the regulation of L-MAG phosphorylation. Evidence was also obtained for the dimerisation of the L-MAG cytoplasmic domain. The S-MAG cytoplasmic domain bound zinc, which induced a change in the surface properties of the protein. The S-MAG cytoplasmic domain was also found to interact directly with tubulin, the core component of microtubules. In conclusion, this study has brought information on the functions of the MAG cytoplasmic domains. The results are complementary with ealier hypotheses on the roles of the MAG isoforms in myelinating glia. While the properties of L-MAG suggest a role as a signaling molecule, a dynamic structural role for S-MAG during myelin formation and maintenance can be envisaged.

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.

Abstract FAP52 (focal adhesion protein, 52 kDa) is a focal adhesion-associated protein composed of a highly α-helical NH2-terminus containing a poorly characterized FCH (Fes/CIP4 homology) domain, unstructured linker region and the COOH-terminal SH3 domain. FAP52 is also known as PACSIN 2 or syndapin II. Together with other PACSINs and syndapins FAP52 shares a common domain architecture. The aim of this study was to characterize FAP52 in structural and functional terms. The function was pursued by identifying binding partners for FAP52, and by overexpressing the recombinant FAP52 in cultured cells. For the structural studies, various physico-chemical methods, such as chemical cross-linking, gel filtration chromatography, circular dichroism and X-ray crystallography were applied. In addition, the histological distribution of FAP52 in chicken tissues was explored. FAP52 binds filamin, a protein that regulates the dynamics of the cytoskeleton by crosslinking actin filaments. The binding site in FAP52 was mapped to the NH2-terminal 184 amino acids, of which the residues 146–184 form the core of the binding. In filamin, the binding site resides in the repeats 15–16 in the rod-like molecule encompassing 24 such repetitive domains. Overexpression of FAP52 or its filamin-binding domain in chicken embryo heart fibroblasts induced the formation of filopodial extensions on the cell surface and reduced the number of focal adhesions, suggesting a role in the organization of the cellular cytoskeleton and in cell adhesion machinery. Experiments utilizing surface plasmon resonance analysis, size exclusion chromatography and chemical cross-linking showed that FAP52 self-associates in vitro and in vivo. The region responsible for the self-association was mapped to the amino acids 146–280, which is predicted to fold into a coiled-coil arrangement. FAP52 was crystallized by using the hanging-drop vapor-diffusion method and ammonium sulfate grid screen. Native dataset was collected from two crystals, which diffracted to 2.8 Å and 2.1 Å resolution. For one form of crystals, phasing was performed using the native dataset and the datasets from two xenon-derivatized crystals. X-ray crystallography studies revealed a dimer in asymmetric unit. Histological and in vitro studies showed that, in liver, FAP52 is preferentially expressed in bile canaliculi. In other tissues, FAP52 showed a specific staining pattern in gut, kidney, brain and gizzard. Together, these data show that FAP52 self-associates in vivo and, probably via its interaction with its binding partner filamin, participates in the organization of the cytoskeletal architecture, especially of the cell surface protrusions, such as filopodia and microvilli of bile canaliculi.

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.