Fiber Cytoskeleton Research Articles

Early life lipid overload in Native American Myopathy is phenocopied by stac3 knockout in zebrafish

Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving musculoskeletal muscle performance, such as restoring the functional integrity of the cytoskeleton. SH3 and cysteine-rich domain 3 (STAC3) are proteins involved in nutrient regulation and are an essential component of the excitation–contraction (EC) coupling machinery for Ca2+ releasing. A mutation in STAC3 causes debilitating Native American Myopathy (NAM) in humans, while loss of this gene in mice and zebrafish (ZF) results in premature death. Clinically, NAM patients demonstrated increased lipids in skeletal muscle, but it is unclear if neutral lipids are associated with altered muscle function in NAM. Using a CRISPR/Cas9 induced stac3-/- knockout (KO) zebrafish model, we determined that loss of stac3 leads to delayed larval hatching which corresponds with muscle weakness and decreased whole-body Ca2+ level during early skeletal development. Specifically, we observed defects in the cytoskeleton in F-actin and slow muscle fibers at 5 and 7 days post-fertilizations (dpf). Myogenesis regulators such as myoD and myf5, mstnb were significantly altered in stac3-/- larvae. These muscle alterations were associated with elevated neutral lipid levels starting at 5 dpf and persisting beyond 7 dpf. Larva lacking stac3 had reduced viability with no larva knockouts surviving past 11 dpf. This data suggests that our stac3-/- zebrafish serve as an alternative model to study the diminished muscle function seen in NAM patients. The data gathered from this new model over time supports a mechanistic view of lipotoxicity as a critical part of the pathology of NAM and the associated loss of function in muscle.

Effects of mutant lamins on nucleo-cytoskeletal coupling in Drosophila models of LMNA muscular dystrophy.

The nuclei of multinucleated skeletal muscles experience substantial external force during development and muscle contraction. Protection from such forces is partly provided by lamins, intermediate filaments that form a scaffold lining the inner nuclear membrane. Lamins play a myriad of roles, including maintenance of nuclear shape and stability, mediation of nuclear mechanoresponses, and nucleo-cytoskeletal coupling. Herein, we investigate how disease-causing mutant lamins alter myonuclear properties in response to mechanical force. This was accomplished via a novel application of a micropipette harpooning assay applied to larval body wall muscles of Drosophila models of lamin-associated muscular dystrophy. The assay enables the measurement of both nuclear deformability and intracellular force transmission between the cytoskeleton and nuclear interior in intact muscle fibers. Our studies revealed that specific mutant lamins increase nuclear deformability while other mutant lamins cause nucleo-cytoskeletal coupling defects, which were associated with loss of microtubular nuclear caging. We found that microtubule caging of the nucleus depended on Msp300, a KASH domain protein that is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Taken together, these findings identified residues in lamins required for connecting the nucleus to the cytoskeleton and suggest that not all muscle disease-causing mutant lamins produce similar defects in subcellular mechanics.

Large Hypertrophy but Unmodified Specific Tension of Single Fibers of Body Builders

IntroductionMuscle hypertrophy is the main outcome of training for body building. Previous studies showed a discrepancy between the functional (force/tension) and structural (cross‐sectional area, CSA) proprieties of leg extensor muscles measured in vivo and the corresponding parameters measured in single muscle fibers [1] [2]. In particular, a puzzling finding from these studies was that of a lower specific tension of skinned fibers of body builders (BB) compared to a control (C) population.In this study we aimed to investigate the possible reasons behind the alleged lower specific tension in single fibers of a strictly diet‐and‐training‐controlled BB population.MethodsFive male body builders (26±4.7 yr; 87.2±9.7 kg; 178.4±7.5 cm) with a training history of at least 5 years were recruited for this study. Five age‐matched recreationally active males (24.6±3.6 yr; 84.4±14.9 kg; 186.4±4.4 cm) were recruited as controls. After ethical approval, biopsies from the vastus lateralis (VL) muscle were collected together with in‐vivo quadriceps maximum isometric voluntary contraction (MVC) and architecture and CSA. Data analysis included (i) single skinned fiber mechanics (n=200 fibers; maximum force, CSA, specific tension and calcium diffusion), (ii) single fiber myosin quantification (n=100 fibers), (iii) slow and fast fiber CSA via histological section analysis. In addition, swelling ratio (skinned fibers CSA/histological CSA) was calculated for each fiber type and for each subject. A general linear mixed model was used to assess statistically significant differences.ResultsQuadriceps MVC and CSA were 64% and 33% higher in BB than C (p<0.05), VL pennation angle (PA) and thickness (MT) were 16% and 15% higher in BB than C (p<0.05). Quadriceps specific force was 25% higher in BB than C (n.s.). Single skinned type 2 CSA and force were 48% and 28% higher in BB than C (p<0.05), while specific tension was 19% lower (p<0.05). Type 1 fibers specific tension was 32% higher in BB than C (p<0.05). Myosin content and calcium diffusion were similar among populations. CSA determined in histological sections was 10% and 27% higher in BB than C (p<0.05) for type1 and 2 fibers, respectively. When type 1 and type 2 fiber tensions were corrected for individual swelling ratio, the difference between BB and C disappeared.ConclusionsAlthough at first sight these findings seem to confirm that in type 2 skinned fibers of BB specific tension is lower than in C, once accounting for fiber swelling, this difference disappears. This is further confirmed by the lack of difference in myosin concentration and calcium diffusion kinetics.In contrast, the results point to the existence of a differential swelling response to the skinning process by BB and C fibers, possibly due to adaptations of the fiber cytoskeleton proteins or specific permeability of the membrane of slow and fast fibers.

The clinical case of limb-girdle muscle dystrophy 2Q associated with myasthenic syndrome and lung damage

Limb-girdle muscle dystrophy 2Q is one of the rarest forms of plectinopathies and is represented by an isolated muscular dystrophic syndrome, according to two previously described literature reports. There are five forms of plectinopathies, including limb-girdle muscle dystrophy 2Q, are caused by mutations in the PLEC gene, the alternative splicing of which determines the synthesis of 9 isoforms of the plectin protein (1, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 3) performing cytolinker function in the neuronal, epithelial and muscle tissue. The article describes the family observation of three sick siblings with the limb-girdle muscle dystrophy 2Q phenotype due to the presence of a new homozygous mutation (NM_201378.3:c.58G>T, NP_958780.1:p.Glu20Ter) in the isoform 1f PLEC revealed by whole-exome sequencing. Clinical, electromyography, visualization and histopathological features of limb-girdle muscle dystrophy 2Q were analyzed in detail. The onset of clinical manifestations in all the described siblings was observed in early childhood with moderate weakness mainly in the pelvic girdle muscles and proximal lower limbs with minimal involvement of the muscles of the shoulder girdle. A distinctive aspect is the stagnation of the myodystrophic process until 20—21 years, followed by the progression and development of episodes of respiratory failure, as well as the formation of rigidity of the cervical, thoracic spine and moderate contracture of the Achilles tendons. Typical features are marked atrophy of paravertebral muscles with the formation of pterygoid scapula and the presence of hypertrophy m. gastrocnemius, m. quadriceps femoris, m. deltoideus and m. triceps brachii. Histopathological examination m. vastus lateralis revealed myodystrophic process without inflammatory infiltration, muscle fiber cytoskeleton disorganization resulted from the plectin loss. Electrocardiography signs of the early repolarization syndrome, focal cardiosclerosis and sinus tachycardia are described. For the first time, involvement in the pathological process of pulmonary tissue in the form of noninfectious bronchiolitis, atelectasis, and the development of the myasthenic syndrome causing episodes of respiratory failure resulted in the death of two described siblings aged 29 and 31 years. Discussed pathogenetic role of PLEC 1f isoform in the development of described syndromes, expands understanding of rare nosology limb-girdle muscle dystrophy 2Q.

Muscular dystrophy meets protein biochemistry, the mother of invention.

Muscular dystrophies result from a defect in the linkage between the muscle fiber cytoskeleton and the basement membrane (BM). Congenital muscular dystrophy type MDC1A is caused by mutations in laminin α2 that either reduce its expression or impair its ability to polymerize within the muscle fiber BM. Defects in this BM lead to muscle fiber damage from the force of contraction. In this issue of the JCI, McKee and colleagues use a laminin polymerization-competent, designer chimeric BM protein in vivo to restore function of a polymerization-defective laminin, leading to normalized muscle structure and strength in a mouse model of MDC1A. Delivery of such a protein to patients could ameliorate many aspects of their disease.

Studying the role of dystrophin-associated proteins in influencing Becker muscular dystrophy disease severity

Becker muscular dystrophy is characterized by a variable disease course. Many factors have been implicated to contribute to this diversity, among which the expression of several components of the dystrophin associated glycoprotein complex. Together with dystrophin, most of these proteins anchor the muscle fiber cytoskeleton to the extracellular matrix, thus protecting the muscle from contraction induced injury, while nNOS is primarily involved in inducing vasodilation during muscle contraction, enabling adequate muscle oxygenation. In the current study, we investigated the role of three components of the dystrophin associated glycoprotein complex (beta-dystroglycan, gamma-sarcoglycan and nNOS) and the dystrophin homologue utrophin on disease severity in Becker patients. Strength measurements, data about disease course and fresh muscle biopsies of the anterior tibial muscle were obtained from 24 Becker patients aged 19 to 66. The designation of Becker muscular dystrophy in this study was based on the mutation and not on the clinical severity. Contrary to previous studies, we were unable to find a relationship between expression of nNOS, beta-dystroglycan and gamma-sarcoglycan at the sarcolemma and disease severity, as measured by muscle strength in five muscle groups and age at reaching several disease milestones. Unexpectedly, we found an inverse correlation between utrophin expression at the sarcolemma and age at reaching disease milestones.

Structure of the cortical cytoskeleton in fibers of postural muscles and cardiomyocytes of mice after 30-day 2-g centrifugation.

Altered external mechanical loading during spaceflights causes negative effects on muscular and cardiovascular systems. The aim of the study was estimation of the cortical cytoskeleton statement of the skeletal muscle cells and cardiomyocytes. The state of the cortical cytoskeleton in C57BL6J mice soleus, tibialis anterior muscle fibers, and left ventricle cardiomyocytes was investigated after 30-day 2-g centrifugation ("2-g" group) and within 12 h after its completion ("2-g + 12-h" group). We used atomic force microscopy for estimating cell's transverse stiffness, Western blotting for measuring protein content, and RT-PCR for estimating their expression level. The transverse stiffness significantly decreased in cardiomyocytes (by 16%) and increased in skeletal muscles fibers (by 35% for soleus and by 29% for tibialis anterior muscle fibers) in animals of the 2-g group (compared with the control group). For cardiomyocytes, we found that, in the 2-g + 12-h group, α-actinin-1 content decreased in the membranous fraction (by 27%) and increased in cytoplasmic fraction (by 28%) of proteins (compared with the levels in the 2-g group). But for skeletal muscle fibers, similar changes were noted for α-actinin-4, but not for α-actinin-1. In conclusion, we showed that the different isoforms of α-actinins dissociate from cortical cytoskeleton under increased/decreased of mechanical load.

Geometrical Micropillars Combined with Chemical Surface Modifications – Independency of Actin Filament Spatial Distribution in Primary Osteoblasts

Cell-biomaterial interactions are strongly affected by topographical and chemical surface characteristics. We found out earlier that geometric titanium (Ti) pillar structures in the micrometer range induce the cells to rearrange their actin cytoskeleton in short fibers solely on the top of the pillars. As a result, cell physiology was hampered concerning collagen I synthesis and spreading capacity. Furthermore, the position-dependent initial cell adhesion strength was declined near the edges. We asked whether these observed cellular effects can be performed only in combination with Ti or occur independently of chemical surface features. In addition, the specific culture conditions, e.g. serum content or influence of gravity, were of interest. Human primary osteoblasts were cultured in Osteoblast Growth Medium with serum containing SupplementMix on pure silicon pillars (5x5x5 μm) or on samples additionally sputtered with Ti (as reference) or gold. To offer the cells ligands for their adhesion receptors, we coated the pillars with collagen I or alternatively with a plasma polymer layer from allylamine. Different from standard culture conditions, the cells were cultured against gravity as well as without serum. The actin cytoskeleton was stained with phalloidin-TRITC after 24 h and analyzed by confocal laser scanning microscopy. Interestingly, on all modifications tested the cell’s actin cytoskeleton was distinctly organized in short fibers on the top of the pillars. Thus, we were able to exclude the influence of (i) the material chemistry (gold, silicon, physical plasma vs. Ti), (ii) the protein deposition on the pillar top and edges, and (iii) the impression caused by gravity.

Structure of cortical cytoskeleton in fibers of mouse muscle cells after being exposed to a 30-day space flight on board the BION-M1 biosatellite

The aim of the work was to analyze changes in the organization of the cortical cytoskeleton in fibers of the mouse soleus muscle, tibialis anterior muscle and left ventricular cardiomyocytes after completion of a 30-day space flight on board the BION-M1 biosatellite (Russia, 2013). The transversal stiffness of the cortical cytoskeleton of the cardiomyocytes and fibers of the skeletal muscles did not differ significantly within the study groups compared with the vivarium control group. The content of beta- and gamma-actin in the membranous fraction of proteins in the left ventricular cardiomyocytes did not differ significantly within all study groups and correlated with the transversal stiffness. A similar situation was revealed in fibers of the soleus muscle and tibialis anterior muscle. At the same time, the content of beta-actin in the cytoplasmic fraction of proteins was found to be decreased in all types of studied tissues compared with the control levels in the postflight group, with lowered beta-actin gene expression rates in the postflight group. After completion of the space flight, the content of alpha-actinin-4 was found to be reduced in the membranous fraction of proteins from the mouse cardiomyocytes, while its content in the cytoplasmic fraction of proteins did not change significantly. Furthermore, gene expression rates of this protein were decreased at the time of dissection (it was started after 13 h after landing). At the same time, the content of alpha-actinin-1 decreased in the membranous fraction and increased in the cytoplasmic fraction of proteins from the soleus muscle fibers.

Cytoskeleton of cortical astrocytes as a target to proline through oxidative stress mechanisms

Hyperprolinemia is an inherited disorder of proline (Pro) metabolism and patients affected by this disease may present neurological manifestations. However, the mechanisms of neural excitotoxicity elicited by hyperprolinemia are far from being understood. Considering the pivotal role of cytoskeletal remodeling in several neurodegenerative pathologies and the potential links between cytoskeleton, reactive oxygen species production and cell death, the aim of the present work was to study the effects of Pro on astrocyte and neuron cytoskeletal remodeling and the possible oxidative stress involvement. Pro induced a shift of actin cytoskeleton in stress fibers together with increased RhoA immunocontent and ERK1/2 phosphorylation/activation in cortical astrocytes. Unlike astrocytes, results evidenced little susceptibility of neuron cytoskeleton remodeling, since Pro-treated neurons presented unaltered neuritogenesis. We observed increased hydrogen peroxide production characterizing oxidative stress together with decreased superoxide dismutase (SOD) and catalase (CAT) activities in cortical astrocytes after Pro treatment, while glutathione peroxidase (GSHPx) activity remained unaltered. However, coincubation with Pro and Trolox/melatonin prevented decreased SOD and CAT activities in Pro-treated astrocytes. Accordingly, these antioxidants were able to prevent the remodeling of the actin cytoskeleton, RhoA increased levels and ERK1/2 phosphorylation in response to high Pro exposure. Taken together, these findings indicated that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Pro and such effects could be mediated, at least in part, by redox imbalance, RhoA and ERK1/2 signaling pathways. The vulnerability of astrocyte cytoskeleton may have important implications for understanding the effects of Pro in the neurotoxicity linked to inborn errors of Pro metabolism.

Organochlorine pesticides induce epithelial to mesenchymal transition of human primary cultured hepatocytes

Persistent organic pollutants (POPs) are a group of organic or chemicals that adversely affect human health and are persistent in the environment. These highly toxic compounds include industrial chemicals, pesticides such as organochlorines, and unwanted wastes such as dioxins. Although studies have described the general toxicity effects of organochlorine pesticides, the mechanisms underlying its potential carcinogenic effects in the liver are not well understood. In this study, we analyzed the effect of three organochlorine pesticides (dichlorodiphenyltrichloroethane, heptachlore and endosulfan) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the epithelial to mesenchymal transition (EMT) in primary cultured human hepatocytes. We found that these compounds modified the hepatocyte phenotype, inducing cell spread, formation of lamellipodia structures and reorganization of the actin cytoskeleton in stress fibers. These morphological alterations were accompanied by disruption of cell–cell junctions, E-cadherin repression and albumin down-regulation. Interestingly, these characteristic features of dedifferentiating hepatocytes were correlated with the gain of expression of various mesenchymal genes, including vimentin, fibronectin and its receptor ITGA5. These various results show that organochlorines and TCDD accelerate cultured human hepatocyte dedifferentiation and EMT processes. These events could account, at least in part, for the carcionogenic and/or fibrogenic activities of these POPs.

Extracellular Adaptations to Altered Force Transmission in Muscle Cells

Interaction between active force generating components and passive extracellular matrix (ECM) force transmitting components provides skeletal muscle with its characteristic biomechanical properties, enabling it to produce work and resist extension. However, the mechanisms behind this “communication” and the cellular structures that enable it are poorly understood. Desmin is an intermediate filament protein integral to the muscle fiber cytoskeleton. It is thought to be involved in stabilizing the contractile apparatus and transmitting force between the cytoskeleton and the ECM. In this study, we investigated the effects of desmin deletion (des-/-) on the composition and mechanical properties of the cellular and extracellular components of muscle.We found that, while des-/- and wt muscles have identical fiber and bundle (fibers+ECM) material properties at birth, these properties diverge becoming disparate with age. Specifically, des-/- fibers become more compliant compared to wt fibers (tangent modulus: 42.8±6 kPa/um and 66.8±7 kPa/um respectively), while des-/- bundles become progressively stiffer compared to wt (tangent modulus: 193.3±19 kPa/um and 129±13 kPa/um respectively). This result suggests that des-/- muscles are chronically altering their ECM. Evidence supporting this hypothesis was provided by significantly increased collagen protein content (des-/-: 8.0±1 ug/mg, wt: 3.4±1 ug/mg), ECM area fraction (des-/-: 15.1±1%, wt: 10.5±1%) and fibrosis-related gene expression patterns in des-/- muscle compared with wt. Additionally, significantly increased numbers of centrally nucleated fibers (des-/-: 5.6±0.5%, wt: 0.7±0.2%) and inflammatory gene expression patterns in des-/- muscle suggest that an increased susceptibility to injury in des-/- fibers may underlie the measured fibrotic response. Thus, in this study, we identify a possible causal relationship between increased cellular compliance, due to reduction in force transmission and stabilization by desmin, and the proliferative response of the extracellular matrix cellular constituents.

Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence-optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

The transforming growth factor‐beta (TGF‐β) mediates acquisition of a mesenchymal stem cell‐like phenotype in human liver cells

Transforming growth factor-beta (TGF-β) mediates several and sometime opposite effects in epithelial cells, inducing growth inhibition, and apoptosis but also promoting an epithelial to mesenchymal transition (EMT) process, which enhances cell migration and invasion. TGF-β plays relevant roles in different liver pathologies; however, very few is known about its specific signaling and cellular effects in human primary hepatocytes. Here we show that TGF-β inhibits proliferation and induces pro-apoptotic genes (such as BMF or BIM) in primary cultures of human fetal hepatocytes (HFH), but also up-regulates anti-apoptotic genes, such as BCL-XL and XIAP. Inhibition of the epidermal growth factor receptor (EGFR), using gefitinib, abrogates the increase in the expression of the anti-apoptotic genes and significantly enhances cell death. Simultaneously, TGF-β is able to induce an EMT process in HFH, coincident with Snail up-regulation and a decrease in E-cadherin levels, cells showing mesenchymal proteins and reorganization of the actin cytoskeleton in stress fibers. Interestingly, these cells show loss of expression of specific hepatic genes and increased expression of stem cell markers. Chronic treatment with TGF-β allows selection of a population of mesenchymal cells with a de-differentiated phenotype, reminiscent of progenitor-like cells. Process is reversible and the mesenchymal stem-like cells re-differentiate to hepatocytes under controlled experimental conditions. In summary, we show for the first time that human hepatocytes may respond to TGF-β inducing different signals, some of them might contribute to tumor suppression (growth inhibition and apoptosis), but others should mediate liver tumor progression and invasion (EMT and acquisition of a stem-like phenotype).

Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle

The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). beta1 integrins are ECM receptors in skeletal muscle, and mutations that affect the alpha7beta1 integrin cause myopathy in humans. In mice, beta1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate beta1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.

Cellular effects of functional unloading and passive strain of soleus muscle in dystrophin-deficient mice

There is a suggestion that dystrophin, a subsarcolemmal protein communicating fiber cytoskeleton to extracellular matrix, participates in signal transduction reflecting the mechanical state of skeletal muscle (mechanotransduction). Recent works indicate the possible signaling role of this protein in the prevention of the activation of proteolytic processes accompanying development of muscle fiber atrophy and in realization of anabolic effects of muscle passive stretching. To assess the role of dystrophin in these processes, the experiment was carried out on two-month old C57 black and mdx (dystrophin-deficient) mice subjected to hind-limb suspension with stretching and without it. Passive stretching results in the partial prevention of atrophy in two muscle fiber types of both C57 black and mdx mice; at the same time, in mdx mice, the slow-to-fast transformation of the soleus muscle fiber type was not observed. Proliferative activity in soleus muscle decreased as a result of hind-limb suspension, but markedly increased during muscle passive stretching. We have found no correlation between the altered dystrophin synthesis and proliferative activity of satellite cells during hind-limb suspension and hind-limb suspension with stretching. Hence, the disturbed dystrophin synthesis retards the atrophy of slow muscle fibers and practically does not affect the stretching preventive action.

Involvement of secreted Aspergillus fumigatus proteases in disruption of the actin fiber cytoskeleton and loss of focal adhesion sites in infected A549 lung pneumocytes.

Aspergillus fumigatus is an opportunistic pathogenic fungus that predominantly infects the respiratory system. Penetration of the lung alveolar epithelium is a key step in the infectious process. The cytoskeleton of alveolar epithelial cells forms the cellular basis for the formation of a physical barrier between the cells and their surroundings. This study focused on the distinct effects of A. fumigatus on the actin cytoskeleton of A549 lung pneumocytes. Of the 3 major classes of cytoskeletal fibers--actin microfilaments, microtubules, and intermediate filaments--only the actin cytoskeleton was found to undergo major structural changes in response to infection, including loss of actin stress fibers, formation of actin aggregates, disruption of focal adhesion sites, and cell blebbing. These changes could be specifically blocked in wild-type strains of A. fumigatus by the addition of antipain, a serine and cysteine protease inhibitor, and were not induced by an alkaline serine protease-deficient strain of A. fumigatus. Antipain also reduced, by approximately 50%, fungal-induced A549 cell detachment from the plates and reduction in viability. Our findings suggest that A. fumigatus breaches the alveolar epithelial cell barrier by secreting proteases that act together to disorganize the actin cytoskeleton and destroy cell attachment to the substrate by disrupting focal adhesions.

F-Actin Is Concentrated in Nonrelease Domains at Frog Neuromuscular Junctions

To gain insight into the role of F-actin in the organization of synaptic vesicles at release sites, we examined the synaptic distribution of F-actin by using a unique synaptic preparation of frog target-deprived nerve terminals. In this preparation, imaging of the synaptic site was unobstructed by the muscle fiber cytoskeleton, allowing for the examination of hundreds of synaptic sites in their entirety in whole mounts. At target-deprived synaptic sites F-actin was distributed in a ladder-like pattern and was colocalized with beta-fodrin. Surprisingly, F-actin stain, which we localized to the nerve terminal itself, did not overlap a synaptic vesicle marker, suggesting that it was concentrated in nonrelease domains of nerve terminals between clusters of synaptic vesicles. These findings suggest that the majority of the presynaptic F-actin is not involved in tethering synaptic vesicles. Instead, the strategic presynaptic positioning of this cytoskeletal meshwork in nonrelease domains of the nerve terminal suggests alternate functions such as restricting synaptic vesicles to release domains, recycling synaptic vesicles, or stabilizing the nerve terminal.

α-Melanocyte-Stimulating Hormone and Endothelin-1 Have Opposing Effects on Melanocyte Adhesion, Migration, and pp125 FAKPhosphorylation

Recent reports show that α-MSH (melanocyte-stimulating hormone) is mitogenic and melanogenic for normal human melanocytes, and that this effect is mediated through binding to the melanocortin receptor (MC1R) and activation of cAMP formation. α-MSH has also been shown to induce changes in cell shape in melanocytes and melanoma cells, particularly increased dendricity, suggesting a potential role for α-MSH in melanocyte–matrix interactions and pigment transfer through reorganization of the melanocyte actin filament cytoskeleton. In this report we show that the potent α-MSH analog (Nle 4, D-Phe 7)–α-MSH (NDP–MSH) induces reorganization of the actin stress fiber cytoskeleton in treated human melanocytes and that this reorganization is associated with increased adhesion to fibronectin (FN). Because most melanocyte growth factors act synergistically on melanocyte mitogenesis, we also sought to determine the effect of the melanocyte mitogen endothelin-1 (ET-1) on the melanocyte actin cytoskeleton, melanocyte adhesion, and melanocyte migration. We show that ET-1, which increases melanocyte migration on FN, has opposite effects on melanocyte adhesion to FN compared with NDP–MSH and that endothelin-1-induced actin reorganization is distinct from that observed following NDP–MSH treatment. Finally, we show that focal adhesion kinase (pp125 FAK), a nonreceptor tyrosine kinase associated with focal contact formation and cell migration, is phosphorylated on tyrosine residues after treatment of melanocytes with ET-1, but not NDP–MSH. These data indicate that while α-MSH and ET-1 act synergistically to modulate melanocyte proliferation, they have opposite effects on melanocyte–matrix interactions.

Presbyopia and velocity of sound in the lens.

The elastic properties of lens matter change with age and this contributes to presbyopia. The changes in elasticity of lens matter could be the result of a change in water and soluble proteins (1) or a change in lens fiber cytoskeleton and membranes (2). If it is caused by (1) then the velocity of sound in the lens should change with age. If it is caused by (2) the velocity of sound in the lens will not change. Using the technique of continuous ultrasonographic biometry, the velocity of sound in clear lenses was measured in vivo in a group of 24 healthy subjects aged 15 to 45 years with a visual acuity of 6/6 or better. In this group maximum accommodative amplitude decreased with age. It was found that, despite the occurrence of presbyopia, the velocity of sound did not change with age. Our results support the hypothesis that age-related changes in lens fiber cytoskeleton and membranes are responsible for the change in elastic properties of lens matter and thus contribute to presbyopia.