Pulmonary endothelial cell (EC) barrier dysfunction and recovery is critical to the pathophysiology of acute respiratory distress syndrome. Cytoskeletal and subsequent cell membrane dynamics play a key mechanistic role in determination of EC barrier integrity. Here, we characterizAQe the actin related protein 2/3 (Arp 2/3) complex, a regulator of peripheral branched actin polymerization, in human pulmonary EC barrier function through studies of transendothelial electrical resistance (TER), intercellular gap formation, peripheral cytoskeletal structures and lamellipodia. Compared to control, Arp 2/3 inhibition with the small molecule inhibitor CK-666 results in a reduction of baseline barrier function (1,241 +/- 53 vs 988 +/- 64 ohm; p < 0.01), S1P-induced barrier enhancement and delayed recovery of barrier function after thrombin (143 +/- 14 vs 93 +/- 6 min; p < 0.01). Functional changes of Arp 2/3 inhibition on barrier integrity are associated temporally with increased intercellular gap area at baseline (0.456 +/- 0.02 vs 0.299 +/- 0.02; p < 0.05) and thirty minutes after thrombin (0.885 +/- 0.03 vs 0.754 +/- 0.03; p < 0.05). Immunofluorescent microscopy reveals reduced lamellipodia formation after S1P and during thrombin recovery in Arp 2/3 inhibited cells. Individual lamellipodia demonstrate reduced depth following Arp 2/3 inhibition vs vehicle at baseline (1.83 +/- 0.41 vs 2.55 +/- 0.46 mm; p < 0.05) and thirty minutes after S1P treatment (1.53 +/- 0.37 vs 2.09 +/- 0.36 mm; p < 0.05). These results establish a critical role for Arp 2/3 activity in determination of pulmonary endothelial barrier function and recovery through formation of EC lamellipodia and closure of intercellular gaps.

Actin stress fibers (SFs) are mechanosensitive structural elements that respond to applied strain to regulate cell morphology, signal transduction, and cell function. The purpose of this dissertation is to elucidate the effects of mechanical stretch on cell mechanobiology via the following three aims. First, a sarcomeric model of SFs was developed to describe the role of actomyosin crossbridge cycling in SF tension regulation and reorientation in response to various modes of stretch. Using model parameters extracted from literature, this model described the dependence of cyclic stretch-induced SF alignment on a two-dimensional (2-D) surface on positive perturbations in SF tension caused by the rate of lengthening, which was consistent with experimental findings. Second, the sarcomeric model was used to predict how stretch-induced pro-inflammatory mechanotransduction depends on the mode of strain application. Together with experimental data, the results indicated that stretch-induced stress fiber alignment, MAPK activations and downstream pro-inflammatory gene expressions are dependent on SF strain rate (and related changes in SF tension) rather than SF turnover. Third, to produce biocompatible materials that are both mechanically resilient under (physiological) load and also mechanosensitive, a novel hybrid engineered tissue was developed that transmits strain stimuli to cells residing in three-dimensional (3-D) collagen microspheres. However, the macroscopic stress is largely borne by a more resilient acellular polyethylene glycol diacrylate (PEGDA) hydrogel supporting the microspheres. Careful analysis indicated that cell alignment occurs prior to significant collagen fibril alignment.

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

<p>    </p> <p>When mammalian eggs are fertilized, they undergo an egg-to-embryo transition during which different egg activation events take place. Egg activation events include the establishment of blocks to polyspermy, which prevent multiple sperm from fertilizing an egg. One of these blocks to polyspermy occurs at the level of the egg plasma membrane (the membrane block to polyspermy). Previous work in our lab provides evidence that the mammalian membrane block to polyspermy is mediated by sperm-induced calcium signaling and the egg’s actomyosin cytoskeleton (McAvey et al., 2002). This thesis research builds upon this foundation, testing hypotheses about two specific effector molecules, one involved in calcium signaling and one with the actin cytoskeleton, and also developing the use of an actin probe for live-cell imaging, with the goal of imaging actin dynamics in eggs undergoing fertilization. Specifically, we examined the calcium effector molecule Ca2+/Calmodulin-dependent-protein kinase IIg (<strong>CaMKII</strong>g), based on previous studies showing that CaMKII plays a role in the membrane block (Gardner et al., 2007) and that the g isoform of CaMKII is necessary and sufficient for eggs to complete meiosis (Backs et al., 2010). We tested the hypothesis that CaMKIIg would mediate the membrane block to polyspermy but found that egg activation driven by expression of a constitutively active form of CaMKIIg was not sufficient to establish the membrane block. Our studies of the actin cytoskeleton focused on the Arp2/3 complex as a candidate. We tested the hypothesis that Arp2/3, which mediates actin filament branching, was involved in membrane block establishment, building on the finding that disruption of actin with the drug cytochalasin D impairs the membrane block (McAvey et al., 2022). These studies used the Arp2/3 inhibitor CK666, predicting that we would see increased sperm incorporation in CK666-treated eggs. However, an assay of sperm incorporation over time indicated that Arp2/3 may not play a significant role in the membrane block to polyspermy, although follow-up studies will be beneficial. Lastly, the actin probe SiR- Actin was assessed for use on oocytes undergoing live-cell imaging during meiosis I and II. Oocytes were treated with differing concentrations of SiR-Actin and live cell imaged while maturing through meiosis I or completing meiosis II. Higher doses and longer exposure to SiR- Actin caused abnormalities in oocytes during meiosis I but not in eggs completing meiosis II. Together, this work sets the stage of a range of future studies into the mammalian membrane block to polyspermy. </p>