Würzburg, Univ., Diss., 2000. / Dateien im PDF-Format. Erscheinungsjahr an der Haupttitelstelle: 1999. Computerdatei im Fernzugriff.

Würzburg, Univ., Diss., 2000. / Dateien im PDF-Format. Erscheinungsjahr an der Haupttitelstelle: 1999. Computerdatei im Fernzugriff.

Würzburg, University, Diss., 2000. / Dateien im PDF-Format. - Erscheinungsjahr an der Haupttitelstelle: 1999.

More than 30 missense mutations in the ACTA2 gene, which encodes α–smooth muscle (α–SM) actin, cause thoracic aortic aneurysms and dissections (TAAD). Aortic cell samples obtained from patients harboring the R256H mutation, the third most common of this group, reveal a lack of α–SM organization. However, the biochemical mechanisms contributing to this disorganization have yet to be elucidated. Biochemical analysis of these mutations is critical to understanding the mechanisms underlying this disease; however, this goal proves difficult due to the inability to obtain diseased aortic tissue, the difficulty in purifying pure mutant actin from smooth muscle tissue, and the absence of a sufficient animal model. The yeast Saccharomyces cerevisiae has a single actin-encoding gene, ACT1, that shares 86% homology with human α–SM actin, making it a viable model system for analysis of TAAD mutations. Regulation of actin function by actin binding proteins is conserved between yeast and humans and plays a key role in cytoskeletal assembly and disassembly events. Two such actin binding proteins are cofilin and Aip1p, which work together to facilitate filament disassembly. Cofilin severs actin filaments, an action enhanced by actin interacting protein 1 (Aip1p) through mechanisms that are not well understood. Normal regulation of filament disassembly is important for cell proliferation and migration. In vivo data obtained by lab colleagues reveal that, in the absence of Aip1p, cells have abnormal cytoskeleton and organelle morphologies and poor growth. This thesis summarizes the investigation into the regulation of R256H mutant actin by Aip1p. S. cerevisiae was used to express R256H actin as the sole actin in the cell, and the effect of the mutation was assessed in vitro. The mutant actin exhibited decreased thermal stability indicative of an effect on monomer integrity. Filament stability was also affected as evidenced by aberrant polymerization kinetics, and high critical concentration and phosphate release rates. While the mutant actin was more sensitive to cofilin-mediated severing, it was less sensitive to disassembly in the presence of Aip1p with incomplete oligomer breakdown at varying Aip1p concentrations. Collectively, these data suggest an alteration in the filament in the presence of the R256H mutation that interferes with proper actin–Aip1p interaction. The biochemical effects observed with this mutant suggest how its presence leads to α–SM cytoskeletal disorganization and TAAD.

Transient Receptor Potential ‘Melastatin’ 7 (TRPM7) is a ubiquitously expressed, non-selective divalent cation channel implicated in diverse cellular functions including actomyosin cytoskeletal remodeling, magnesium homeostasis, and anoxic neuronal death. The present study investigates the role of TRPM7 in modulating neuronal morphology and regulating neuronal cytoskeletal dynamics after anoxia. Overexpression of GFP-tagged TRPM7 in neuronal cultures caused a stunted morphology with fewer neurite branches than controls, suggesting that TRPM7 regulates the neuronal cytoskeleton during dendritic outgrowth. I have discovered that TRPM7 may regulate morphology via activation of cofilin-1 (an actin binding protein). I found that TRPM7-dependent cofilin activation during anoxia mediated neuronal death. Overall my work reveals a novel link between anoxia-induced TRPM7 activity and cofilin activation, which likely contributes to neurodegeneration after ischemia.

Transient Receptor Potential ‘Melastatin’ 7 (TRPM7) is a ubiquitously expressed, non-selective divalent cation channel implicated in diverse cellular functions including actomyosin cytoskeletal remodeling, magnesium homeostasis, and anoxic neuronal death. The present study investigates the role of TRPM7 in modulating neuronal morphology and regulating neuronal cytoskeletal dynamics after anoxia. Overexpression of GFP-tagged TRPM7 in neuronal cultures caused a stunted morphology with fewer neurite branches than controls, suggesting that TRPM7 regulates the neuronal cytoskeleton during dendritic outgrowth. I have discovered that TRPM7 may regulate morphology via activation of cofilin-1 (an actin binding protein). I found that TRPM7-dependent cofilin activation during anoxia mediated neuronal death. Overall my work reveals a novel link between anoxia-induced TRPM7 activity and cofilin activation, which likely contributes to neurodegeneration after ischemia.

Ulm, Univ., Diss., 2008.

Indiana University-Purdue University Indianapolis (IUPUI) / Our ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.

No description available.

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.