<p>The ability of cells to migrate is of fundamental importance to a diverse array of biological processes, both physiological and pathological, such as development, the immune response and cancer cell metastasis, to name a few. The process of cell movement is a complicated cycle of coordinated steps involving dynamic and precise rearrangement of the actin-myosin cytoskeleton. As a critical component of the migration machinery, the molecular motor protein nonmuscle myosin II (myosin II) has long been a subject of scientific inquiry. It is now generally accepted that the contractile forces generated by myosin II contribute directly or indirectly to every step in migration. Interestingly, three isoforms of myosin II (myosin IIA, IIB and IIC) have been identified, and although each isoform performs the same basic molecular functions, recent findings suggest that the different myosin II isoforms make unique contributions to the motile process. In this dissertation work I used RNA interference technology to specifically deplete cells of myosin IIA and IIB in order to characterize the distinct migration phenotypes associated with loss-of-function of each individual isoform. Surprisingly, I found that the two myosin II isoforms perform not only distinct but opposing functions in cell migration, with myosin IIA and IIB normally inhibiting and facilitating proper cell movement, respectively. Furthermore, using pharmacological and microscopy techniques, I investigated the cellular mechanisms allowing for isoform-specific function. My results provide evidence for at least two isoform-specific regulatory mechanisms, namely selectivity in signaling pathways and subcellular distribution. A particularly significant finding is the identification of the different assembly properties of myosin IIA and IIB as the key element responsible for directing isoform-distinct distribution. Together the data presented herein represent a considerable advance in our understanding of the distinct functions and regulation of myosin II in cell motility.</p> / Dissertation

Lung tumors secrete multiple factors that contribute to cachexia, a severe wasting syndrome that includes loss of muscle mass, weakness, and fatigue. 80% of advanced lung cancer patients experience cachexia, which cannot be reversed by nutritional interventions, diminishes response to and tolerance of cancer treatments, and increases morbidity and mortality. Despite a multitude of clinical trials, there are currently no approved treatments. This deficiency suggests that not all of the factors that contribute to cachexia have been identified. Cancer is frequently accompanied by an increase in cyclooxygenase-2 (COX-2), a hallmark of inflammation. Clinical trials for COX-2 inhibitors have resulted in restoration of muscle mass and decreased fatigue. Along with loss of myofibrillar proteins, cachexia also induces mitochondrial dysfunction, which contributes to fatigue. The amelioration of fatigue by COX-2 inhibition suggests possible alterations to mitochondrial function. We hypothesized that there were unidentified tumor-derived factors that contribute to cachectic wasting and fatigue. Treatment of C2C12 myotubes with Lewis lung cancer-conditioned media (LCM) resulted in increased COX-2 content, myosin loss, and mitochondrial dysfunction. Mass spectrometry revealed 158 confirmed proteins in LCM. We focused on extracellular 14-3-3 proteins because they bind and regulate over 200 known partners. We found that depletion of extracellular 14-3-3 proteins diminished myosin content. CD13, an aminopeptidase, is the proposed receptor for 14-3-3 proteins. Inhibiting aminopeptidases with Bestatin also reduced myosin content. LCM treatment decreased basal and ATP-related mitochondrial respiration, caused a transient rise in reactive oxygen species (ROS), and increased 4-Hydroxynonenal (4-HNE) in both cytosolic and mitochondrial fractions of cell lysates. COX-2 inhibition did not spare myosin content in LCM-treated myotubes, but did alter mitochondrial respiration and cytosolic oxidant levels. Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines, signaling via aminopeptidases to help maintain muscle mass. We elucidated how LCM alters mitochondrial electron flow, and increases oxidative damage by ROS and 4-HNE. Although successful in clinical trials, COX-2 inhibitors do not appear to spare muscle mass by directly working on skeletal muscle, but did alter mitochondrial function.

Introduction: The extraocular muscles (EOMs) are considered a separate class of skeletal muscle, allotype. Myosin is the major contractile protein in muscle. The myosin heavy chain (MyHC) isoforms are the best molecular markers of functional heterogeneity of muscle fibers. The relaxation rate, reflects the rate at which Ca2+ is transported back into the sarcoplasmic reticulum (SR) mostly by SR Ca2+ATPase (SERCA). Myosin binding protein C (MyBP-C), plays a physiological role in regulating contraction. The laminins (Ln) are the major non-collagenous components of the basement membrane (BM) surrounding muscle fibers and are important for muscle fiber integrity. Methods: Adult human EOMs were studied with SDS-PAGE, immunoblots and immunocytochemistry, the latter with antibodies against six MyHC, 2 SERCA, 2 MyBP-C and 8 laminin chain isoforms. The capillary density was also determined. Results: Most fibers contained a mixture of MyHC isoforms. Three major groups of fibers could be distinguished. Fast fibers that stained with anti-MyHCIIa, slow fibers that stained with anti-MyHCI and MyHCeompos/MyHCIIaneg-fibers that stained with neither of these antibodies but with anti-MyHCI+IIa+eom and anti-MyHCeom. A majority of the fibers contained both SERCA1 and 2 whereas 1% were unstained with both antibodies. Biochemically SERCA2 was more abundant than SERCA1. MyBP-Cfast was not present in the EOMs and MyBP-Cslow was only detected immunocytochemically. The extrasynaptical BM of the EOM muscle fibers contained Lna2, b1, b2, g1, a4 and a5 chains. The capillary density in the EOMs was very high (1050 +/-190 capillaries/mm2) and significantly (p&lt;0.05) higher in the orbital than in the global layer. Conclusions: The co-existence of complex mixtures of several crucial protein isoforms provide the human EOMs with a unique molecular portfolio that a) allows a highly specific fine-tuning regime of contraction and relaxation, and b) imparts structural properties that are likely to contribute to protection against certain neuromuscular diseases.

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science

The purpose of this investigation was to examine the effects of Multiple Sclerosis (MS) on the structural and functional characteristics of skeletal muscle. More specifically, we analyzed the myosin heavy chain (MHC) and fiber type distribution of the vastus lateralis, measured single fiber cross sectional area (CSA), and determined the isokinetic and isotonic strength of the knee extensor muscles. Six sedentary subjects with MS (age: 44 ± 2 yrs) and six sedentary gender-matched controls (age: 46 ± 4) were evaluated. EachMS subject was rated on the Kurtzke's Expanded Disability Status Scale (EDSS) and performed an 8-meter walk test to determine gait speed. Furthermore, the spasticity of the knee extensors was evaluated in each MS subject and weekly energy expenditure was estimated using the Yale Physical Activity Survey. Concentric and eccentric isokinetic strength of the right knee extensors (left in one MS subject) was determined at 60 and 180°/sec and a bilateral isotonic one-repetition maximum (1-RM) was evaluated in eachsubject. Muscle biopsies were taken from the right vastus lateralis (left in one MS subject) and individual fibers were dissected from these samples. Fibers were submitted to SDSPAGE with silver staining to determine MHC expression. Densitometry was performed on MHC hybrid fibers to determine the degree of co-expression. An additional section ofthe biopsy was stained for mATPase activity and further analyzed for single fiber CSA and fiber type. The mean EDSS score for the MS subjects was 5.4 ± 0.6 (range 3.5-6.5) and MS patients were slower than controls (p < 0.05) on the walk-test. AshworthSpasticity Scores ranged from 0 - 2. No differences were noted in weekly energy expenditure. The controls were 45 and 56% stronger than the MS group at isokinetic concentric velocities of 60 and 180°/sec (p < 0.05), respectively. The isotonic 1-RM andthe eccentric isokinetic contractions were not different between the two groups. There were no differences noted in any of the MHC isoforms or percentage of hybrid fibers. Furthermore, mATPase fiber type distribution and single fiber CSA were not different between the groups. There was a greater proportion of MHC IIx dominant MHC IIa/IIx fibers in the MS groups (p < 0.05). Multiple Sclerosis appears to result in large strengthdeficits, when compared to healthy individuals. Based on our findings, these strength differences cannot be explained by alterations in MHC/fiber type expression or decreases in fiber CSA. / School of Physical Education

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

Running title: Climbing fibers in myosin Va mutants

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.

Acute quadriplegic myopathy (AQM), or critical illness myopathy, is a common debilitating acquired disorder in critically ill intensive care unit (ICU) patients characterized by generalized muscle wasting and weakness of limb and trunk muscles. A preferential loss of the thick filament protein myosin is considered pathognomonic of this disorder, but the myosin loss is observed relatively late during the disease progression. In attempt to explore the potential role of factors considered triggering AQM in sedated mechanically ventilated (MV) ICU patients, we have studied the early effects, prior to the myosin loss, of neuromuscular blockade (NMB), corticosteroids (CS) and sepsis separate or in combination in a porcine experimental ICU model. Specific interest has been focused on skeletal muscle gene/protein expression and regulation of muscle contraction at the muscle fiber level. This project aims at improving our understanding of the molecular mechanisms underlying muscle specific differences in response to the ICU intervention and the role played by the different triggering factors. The sparing of masticatory muscle fiber function was coupled to an up-regulation of heat shock protein genes and down-regulation of myostatin are suggested to be key factors in the relative sparing of masticatory muscles. Up-regulation of chemokine activity genes and down-regulation of heat shock protein genes play a significant role in the limb muscle dysfunction associated with sepsis. The effects of corticosteroids in the development of limb muscle weakness reveals up-regulation of kinase activity and transcriptional regulation genes and the down-regulation of heat shock protein, sarcomeric, cytoskeletal and oxidative stress responsive genes. In contrast to limb and craniofacial muscles, the respiratory diaphragm muscle responded differently to the different triggering factors. MV itself appears to play a major role for the diaphragm muscle dysfunction. By targeting these genes, future experiments can give an insight into the development of innovative treatments expected at protecting muscle mass and function in critically ill ICU patients.