Calcific aortic valve sclerosis is characterized by focal lesions in the valve leaflet. These lesions are rich in myofibroblasts that express α-SMA and cause fibrosis. Lesions tend to occur in regions of the leaflet that are subjected to large bending loads, suggesting a mechanobiological basis for myofibrogenic differentiation and valve pathogenesis. In this thesis, a bioreactor was developed to study the effect of physiological loading on myofibrogenic differentiation of valve interstitial cells. Cyclic loading of native porcine aortic valve leaflets ex vivo resulted in increased α-SMA expression, predominantly in the fibrosa and spongiosa (similar to sclerotic leaflets). Cofilin, an actin-binding protein, was also upregulated by loading, suggesting it plays a role in mechanically-induced myofibrogenesis. Similarly, loading of a tissue engineered aortic valve leaflet model resulted in increased α-SMA transcript and protein expression. These data support an integral role for mechanical stimuli in myofibrogenic differentiation and sclerosis in the aortic valve.

The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

Traumatic brain injury (TBI) is a relatively unknown disease considering the tens of millions of people affected around the world each year. Many TBI patients die from their injuries and survivors often suffer from life-long disabilities. The primary injury initiates a variety of cellular and molecular processes that are both beneficial and detrimental for the brain, but that are not fully understood. The focus of this thesis has been to study the role of astrocytes in clearance of dead cells after TBI and to identify injury specific proteins that may function as biomarkers, by using cell cultures, animal models and in cerebrospinal fluid (CSF) from TBI patients. The result demonstrates a new function in that astrocytes, the most numerous cell type in the brain, engulf dead cells after injury both in cell cultures and in adult mice and thereby save neurons from contact-induced apoptosis. Astrocytes are effective phagocytes, but degrade the ingested dead cells very slowly. Moreover, astrocytes express the lysosome-alkalizing proteins Rab27a and Nox2 as well as major histocompatibility complex class II, the receptors on which antigens are being presented. By lowering the pH of the lysosomes with acidic nanoparticles, the degradation increases, but the astrocytes still remained less effective than macrophages. Taken together, the data indicates that the low acidification in astrocytes can preserve antigens and that astrocytes may be able to activate T cells. The expression and secretion of injury-specific proteins was studied in a cell culture model of TBI by separate mass spectrometry analysis of cells and medium. Interestingly, close to 30 % of the injury-specific proteins in medium are linked to actin, for example ezrin of the ezrin/radixin/moesin (ERM) protein family. Ezrin, but none of the other ERM proteins or actin, is actively secreted after injury. Extracellular ezrin also increases in CSF in response to experimental TBI in rats and is present in CSF from TBI patients, indicating that ezrin is a potential biomarker for TBI.

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The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

Cell-substrate interactions influence various cellular processes such as morphology, motility, proliferation and differentiation. Actin dynamics within cells have been shown to be influenced by substrate stiffness, as NIH 3T3 fibroblasts grown on stiffer substrates tend to exhibit more prominent actin stress fiber formation. Circular dorsal ruffles (CDRs) are transient actin-rich ring-like structures within cells, induced by various growth factors, such as the platelet-derived growth factor (PDGF). CDRs grow and shrink in size after cells are stimulated with PDGF, eventually disappearing ten of minutes after stimulation. As substrate stiffness affect actin structures and cell motility, and CDRs are actin structures which have been previously linked to cell motility and macropinocytosis, the role of substrate stiffness on the properties of CDRs in NIH 3T3 fibroblasts and how they proceed to affect cell behavior is investigated. Cells were seeded on Poly-dimethylsiloxane (PDMS) substrates of various stiffnesses and stimulated with PDGF to induce CDR formation. It was found that an increase in substrate stiffness increases the lifetime of CDRs, but did not affect their size. A mathematical model of the signaling pathways involved in CDR formation is developed to provide insight into this lifetime and size dependence, and is linked to substrate stiffness via Rac-Rho antagonism. CDR formation did not affect the motility of cells seeded on 10 kPa stiff substrates, but is shown to increase localized lamellipodia formation in the cell via the diffusion of actin from the CDRs to the lamellipodia. To further probe the influence of cell-substrate interactions on cell behavior and actin dynamics, a two dimensional system which introduces a dynamically changing, reversible and localized substrate stiffness environment is constructed. Cells are seeded on top of thin PDMS nano-membranes, and are capable of feeling through the thin layer, experiencing the stiffness of the polyacrylamide substrates below the nano-membrane. The membranes are carefully re-transplanted on top of other polyacrylamide substrates with differing stiffnesses. This reversible dynamic stiffness system is a novel approach which would help in the investigation of the influence of reversible dynamic stiffness environments on cell morphology, motility, proliferation and differentiation in various cells types.

Staphylococcus aureus are Gram-positive bacteria that adhere to the extracellular matrix of susceptible host cells to initiate infection and induce a signal transduction pathway that includes PI3K causing the disruption of cytoskeletal elements within the cytosol. Confocal microscopy was applied to visualize actin within human umbilical vein endothelial cells (HUVEC) to discern behavior during infection. HUVEC lysates were analyzed through immunoprecipitation and Western blot analysis to determine the isoforms of PI3K present in HUVECs. Infection experiments and confocal microscopy reveal a time dependent disruption of actin and a dose dependent decrease in infection when HUVECs are treated with the PI3K inhibitor LY294002. Results of Western blot analysis reveal a distinct band corresponding to the pl l0a isoform of PI3K in HUVECs. These studies taken together suggest that PI3K is involved in the signal transduction pathway induced by the infection of HUVECs by S. aureus, and that infection causes the disruption of cytoskeletal actin fibers. / Department of Biology

No description available.

Congenital myopathies are a rare and heterogeneous group of diseases. They are primarily characterised by skeletal muscle weakness and disease-specific pathological features. They harshly limit ordinary life and in severe cases, these myopathies are associated with early death of the affected individuals. The congenital myopathies investigated in this thesis are nemaline myopathy and myofibrillar myopathy. These diseases are usually caused by missense mutations in genes encoding myofibrillar proteins, but the exact mechanisms by which the point mutations in these proteins cause the overall weakness remain mysterious. Hence, in this thesis two different nemaline myopathy-causing actin mutations and one myofibrillar myopathy-causing myosin-mutation found in both human patients and mouse models were used to investigate the cascades of molecular and cellular events leading to weakness. I performed a broad range of functional and structural experiments including skinned muscle fibre mechanics, small-angle X-ray scattering as well as immunoblotting and histochemical techniques. Interestingly, according to my results, point mutations in myosin and actin differently modify myosin binding to actin, cross-bridge formation and muscle fibre force production revealing divergent mechanisms, that is, gain versus loss of function (papers I, II and IV). In addition, one point mutation in actin appears to have muscle-specific effects.  The presence of that mutant protein in respiratory muscles, i.e. diaphragm, has indeed more damaging consequences on myofibrillar structure than in limb muscles complexifying the pathophysiological mechanisms (paper II). As numerous atrophic muscle fibres can be seen in congenital myopathies, I also considered this phenomenon as a contributing factor to weakness and characterised the underlying causes in presence of one actin mutation. My results highlighted a direct muscle-specific up-regulation of the ubiquitin-proteasome system (paper III). All together, my research work demonstrates that mutation- and muscle-specific mechanisms trigger the muscle weakness in congenital myopathies. This gives important insights into the pathophysiology of congenital myopathies and will undoubtedly help in designing future therapies.

<p>Mutations in myosin VIIa (MyoVIIa), an unconventional myosin, have been shown to cause Usher Syndrome Type 1B in humans. Usher Syndrome Type 1B is characterized by congenital sensorineural deafness, vestibular dysfunction and pre-pubertal onset of <italic>retinitis pigmentosa</italic>. Mouse model studies show that sensorineural deafness and vestibular dysfunction in MyoVIIa mutants is caused by disruption in the structure of microvilli-like projections (stereocilia) of hair cells in the cochlea and vestibular organ. MyoVIIa has also been shown to affect adaptation of mechanoelectrical transduction channels in stereocilia. </p><p>In <italic>Drosophila melanogaster</italic> mutations in MyoVIIa encoded by <italic>crinkled (ck)</italic> cause defects in hair and bristle morphogenesis and deafness. Here we study the formation of bristles and hairs in <italic>Drosophila melanogaster</italic> to investigate the molecular basis of ck/MyoVIIa function and its regulation. We use live time-lapse confocal microscopy and genetic manipulations to investigate the requirement of ck/MyoVIIa function in various steps of morphogenesis of hairs and bristles. Here we show that null or near null mutations in ck/MyoVIIa lead to the formation of 8-10 short and thin hairs (split hairs) per epithelial cell that are likely the result of the failure of association of hair-actin bundles that in wild-type cells come together to form a single hair.</p><p>The myosin super family of motor proteins is divided into 17 classes by virtue of differences in the sequence of their motor domain, which presumably affect their physiological functions. In addition, substantial variety in the overall structure of their tail plays an important role in the differential regulation of myosin function. In this study we show that ck/MyoVIIa, that has two MyTH4 FERM domains in its tail separated by an SH3 domain, requires both MyTH4 FERM repeats for efficient association of hair-actin bundles to form hairs. We also show that the "multiple hair" phenotype of over-expression of ck/MyoVIIa requires both MyTH4 FERM domain function but not the tail-SH3 domain. We further demonstrate that the tail-SH3 domain of ck/MyoVIIa plays a role in keeping actin bundles, which run parallel to the length of the growing bristle, separate from each other. Our data also suggests that the tail-SH3 domain plays a role in the association of the actin filament bundles with the membrane and regulates F-actin levels in bristles.</p><p>We further demonstrate that over-expression of <italic>Quail</italic> (villin) can rescue the hair elongation defects seen in ck/MyoVIIa null or near null mutants but does not rescue the split hair defects. We show that over-expression of <italic>Alpha-actinin-GFP</italic>, another actin bundling protein, phenocopies the multiple hair phenotype of ck/MyoVIIa over-expression. Over-expression of <italic>Alpha-actinin-GFP</italic> in a ck/MyoVIIa null or near null background shows that <italic>Alpha-actinin-GFP</italic> cannot rescue the split or short hair phenotype of ck/MyoVIIa loss-of-function. However, cells over-expressing <italic>Alpha-actinin-GFP</italic> in a ck/MyoVIIa null or near null background have more than the normal 8-10 split hairs, suggesting that <italic>Alpha-actinin-GFP</italic> over-expression causes the formation of more than the normal complement of hair-actin bundles per cell, resulting in a multiple hair phenotype. We show that <italic>Twinfilin</italic>, an actin monomer sequestering protein implicated in negatively regulating F-actin bundle elongation in stereocilia in a MyoVIIa-dependent manner, is required for F-actin bundle stability. </p><p>In addition, we use yeast two-hybrid strategies to identify <italic>Slam</italic> as a protein that directly binds to ck/MyoVIIa. We show that <italic>Slam</italic>, a novel membrane-associated protein, likely functions to regulate ck/MyoVIIa function during hair and bristle morphogenesis. We show that over-expression of <italic>Slam</italic> and loss-of-function mutations in <italic>Slam</italic> phenocopy ck/MyoVIIa loss-of-function split and short hair phenotype. We also show that disruption of <italic>Slam</italic> and <italic>RhoGEF2</italic> association causes split hair defects similar to ck/MyoVIIa loss-of-function phenotype suggesting that Slam probably regulates ck/MyoVIIa function via <italic>RhoGEF2</italic>.</p><p>Together our results show that ck/MyoVIIa plays an important role in regulating the actin cytoskeleton that underlies actin-based cellular protrusions like hairs and bristles.</p> / Dissertation