Localization of sarcoglycan, neuronal nitric oxide synthase, beta-dystroglycan, and dystrophin molecules in normal skeletal myofiber: triple immunogold labeling electron microscopy.

In order to investigate the mode of existence of the sarcoglycan complex, neuronal nitric oxide synthase (nNOS), beta-dystroglycan, and dystrophin in the normal skeletal myofiber, we examined the ultrastructural localization and mutual spatial relationship of nNOS, beta-dystroglycan, dystrophin, and the individual components of the sarcoglycan complex by using triple immunogold labeling electron microscopy. Each molecule of alpha-, beta-, gamma- and delta-sarcoglycans is located intracellularly or extracellularly near the muscle plasma membrane mostly in accordance with the sarcoglycan antigenic sites against which the antibodies were generated. The association of different two and/or three sarcoglycan molecules out of alpha-, beta-, gamma- and delta-sarcoglycan molecules was frequently observed. Each molecule of nNOS, beta-dystroglycan, and dystrophin was ultrastructurally noted along the cell surface of normal skeletal myofibers. Moreover, the close relation of a sarcoglycan molecule with beta-dystroglycan and dystrophin, and the association of nNOS with dystrophin were also confirmed ultrastructurally. Thus, this study demonstrated that the constituting molecules of the sarcoglycan complex, nNOS, beta-dystroglycan, and dystrophin existed in the form of a cluster at the normal muscle plasma membrane. The association of nNOS with dystrophin and its associated glycoproteins may form a macromolecular signaling complex at the muscle plasma membrane.     

Dystrophin and utrophin: genetic analyses of their role in skeletal muscle

Since the identification of dystrophin as the causitive factor in Duchenne muscular dystrophy, there has been substantial progress in understanding the functions and interactions of this protein. Dystrophin has been shown to interact with a group of peripheral- and trans-membrane proteins known as the dystrophin-associated protein complex (DAPC) and mutations in some of the members of this complex have been shown to account for other forms of muscular dystrophy. This review summarizes the experiments using transgenic and knockout mouse models that have defined the roles of dystrophin, and the dystrophin-related protein utrophin at the skeletal muscle membrane and at the neuromuscular junction. These studies are presented in the context of other known interactions at the muscle membrane. Studies of the dystrophin-deficient mdx mouse have lead to a greater understanding of the human disease. Knockouts and transgenics of utrophin have shown this protein to be sufficient to functionally compensate for dystrophin. Dystrophin transgenic mice combined with the mdx mouse have been used to study the function of specific domains of the dystrophin protein. Together these animal models have led to a delineation of protein functions and localization patterns that will be useful for the generation of potential therapies for DMD.     

Regulation and functional significance of utrophin expression at the mammalian neuromuscular synapse

Duchenne muscular dystrophy (DMD) is caused by the absence of full-length dystrophin molecules in skeletal muscle fibers. In normal muscle, dystrophin is found along the length of the sarcolemma where it links the intracellular actin cytoskeleton to the extracellular matrix, via the dystrophin-associated protein (DAP) complex. Several years ago, an autosomal homologue to dystrophin, termed utrophin, was identified and shown to be expressed in a variety of tissues, including skeletal muscle. However, in contrast to the localization of dystrophin in extrajunctional regions of muscle fibers, utrophin preferentially accumulates at the postsynaptic membrane of the neuromuscular junction in both normal and DMD adult muscle fibers. Since it has recently been suggested that the upregulation of utrophin might functionally compensate for the lack of dystrophin in DMD, considerable interest is now directed toward the elucidation of the various regulatory mechanisms presiding over expression of utrophin in normal and dystrophic skeletal muscle fibers. In this review, we discuss some of the most recent data relevant to our understanding of the impact of myogenic differentiation and innervation on the expression and localization of utrophin in skeletal muscle fibers.     

The spectrin-based skeleton at the postsynaptic membrane of the neuromuscular junction

Membrane skeletons, in particular the spectrin-based skeleton, are thought to participate in the organization of specialized membrane domains by restricting integral proteins to specific membrane sites. In the neuromuscular junction, discrete isoforms of spectrin and ankyrin, the peripheral protein that links spectrin to the membrane, colocalize with voltage-dependent sodium channels and N-CAM at the troughs of the postsynaptic membrane folds. Moreover, beta-spectrin, N-CAM, and sodium channels become clustered at the endplate during a period of time coincident with postsynaptic fold formation and synapse maturation. These observations suggest a role of the spectrin skeleton in directing and maintaining postsynaptic accumulations of sodium channels and N-CAM. In addition, the coexistence of spectrin and dystrophin at the troughs of the junctional folds raises the question of their respective functions in this membrane domain, where both cytoskeletal proteins have the potential to associate with sodium channels via ankyrin and syntrophin, respectively. Possible scenarios are discussed here with respect to accumulating evidence from studies of assembly of similar membrane domains in neurons.     

Sarcoglycans in muscular dystrophy

Muscular dystrophy is a heterogeneous genetic disease that affects skeletal and cardiac muscle. The genetic defects associated with muscular dystrophy include mutations in dystrophin and its associated glycoproteins, the sarcoglycans. Furthermore, defects in dystrophin have been shown to cause a disruption of the normal expression and localization of the sarcoglycan complex. Thus, abnormalities of sarcoglycan are a common molecular feature in a number of dystrophies. By combining biochemistry, molecular cell biology, and human and mouse genetics, a growing understanding of the sarcoglycan complex is emerging. Sarcoglycan appears to be an important, independent mediator of dystrophic pathology in both skeletal muscle and heart. The absence of sarcoglycan leads to alterations of membrane permeability and apoptosis, two shared features of a number of dystrophies. beta-sarcoglycan and delta-sarcoglycan may form the core of the sarcoglycan subcomplex with alpha- and gamma-sarcoglycan less tightly associated to this core. The relationship of epsilon-sarcoglycan to the dystrophin-glycoprotein complex remains unclear. Animals lacking alpha-, gamma- and delta-sarcoglycan have been described and provide excellent opportunities for further investigation of the function of sarcoglycan. Dystrophin with dystroglycan and laminin may be a mechanical link between the actin cytoskeleton and the extracellular matrix. By positioning itself in close proximity to dystrophin and dystroglycan, sarcoglycan may function to couple mechanical and chemical signals in striated muscle. Sarcoglycan may be an independent signaling or regulatory module whose position in the membrane is determined by dystrophin but whose function is carried out independent of the dystrophin-dystroglycan-laminin axis.     

Association of neuronal nitric oxide synthase (nNOS) with alpha1-syntrophin at the sarcolemma.

alpha1-syntrophin is a PDZ-containing dystrophin-associated protein, expressed predominantly in striated muscle and brain. alpha1-syntrophin null mice generated by gene targeting technique showed no overt muscular dystrophic phenotype. Though other dystrophin-associated proteins were localized at the sarcolemma, neuronal nitric oxide synthase (nNOS) was selectively lost from the membrane fraction but remained in the cytoplasm. Thus, the alpha1-syntrophin null mice are useful in the elucidation of the functional importance of nNOS targeting at the sarcolemma. In addition, the mice would facilitate identification of other signaling molecules, which are targeted to dystrophin complex via interaction with alpha1-syntrophin.     

Junctional complexes and cell polarity in the urinary tubule

In this review, we demonstrate how differentiated membrane domains can be detected in epithelial cells using conventional light and electron microscopy, freeze-fracture electron microscopy and the immunoand cytochemical detection of membrane components. Using specific examples from the kidney, we show how the polarized insertion of these components into either apical or basolateral plasma membrane regions on either side of the tight junction barrier is related to specific functions of principal and intercalated cells in the collecting duct. In addition, distinct basal and lateral membrane domains have been revealed in some cells that are maintained in the absence of a tight junctional barrier in the plane of the membrane. This suggests that other factors, possibly related to cytoskeletal elements, may be involved in the functional segregation of these membrane areas. We propose that epithelial cell plasma membranes should be subdivided into apical, lateral and basal regions, and that the term “basolateral” may be an oversimplification.     

Can scanning near‐field optical microscopy be compared with confocal laser scanning microscopy? A preliminary study on α‐sarcoglycan and β1D‐integrin in human skeletal muscle

The dystrophin–glycoprotein complex and the vinculin–talin–integrin system constitute, together a protein machinery, called costameres. The dystrophin–glycoprotein complex contains, among other proteins, also dystrophin and the sarcoglycans subcomplex, proteins playing a key role in the pathogenesis of many muscular dystrophies and linking the cytoplasmic myofibrillar contractile elements to the signal transducing molecules of the extracellular matrix, also providing structural support to the sarcolemma. The vinculin–talin–integrin system connects some components of the extracellular matrix with intermediate filaments of desmin, forming transverse bridges between Z and M lines. In our previous reports we always studied these systems by confocal laser scanning microscopy (CLSM). In this paper we report on the first applications of optical near‐field fluorescence microscopy to the spatial localization of α‐sarcoglycan and β1D‐integrin in human skeletal muscle fibres in order to better compare and test the images obtained with conventional CLSM and with scanning near‐field optical microscopy (SNOM). In addition, the analysis of the surface morphology, and the comparison with the fluorescence map is put forward and analyzed for the first time on human muscle fibres. In aperture‐SNOM the sample is excited through the nanometre‐scale aperture produced at the apex of an optical fibre after tapering and subsequent metal coating. The acquisition of the topography map, simultaneously to the optical signal, by SNOM, permits to exactly overlap the fluorescence images obtained from the two consecutive scans needed for the double localization. Besides, the differences between the topography and the optical spatial patterns permit to assess the absence of artefacts in the fluorescence maps. Although the SNOM represented a good method of analysis, this technique remains a complementary method to the CLSM and it can be accepted in order to confirm the hypothesis advanced by CLSM.     

Atomic force microscopy demonstration of cytoskeleton instability in mouse erythrocytes with dematin-headpiece and β-adducin deficiency

The pattern of disassembly of the cytoskeletal network of murine erythrocytes with deficiency of either dematin-headpiece or β-adducin or both proteins were investigated using atomic force microscopy. A heterogeneous complex structure with fine filament features and coarse features was observed in the cytoskeleton of wild type mouse erythrocytes, whereas a significant amount of rearrangement and aggregation occurred in the mutants, particularly in the cells carrying double gene mutations. These results are consistent with the cellular and biochemical phenotype of the mutant cell membranes as being more fragile due to weakened vertical connections with the plasma membrane.     

Imaging molecular structure and physiological function of gap junctions and hemijunctions by multimodal atomic force microscopy

Gap junctions are specialized plasma membrane structures that join neighboring cells via specialized intercellular ion channels (hemichannels) and provide a direct pathway for cell-cell communication. They presumably mediate regulation of growth, transmission of developmental signals, coordination of muscle contraction, and maintenance of metabolic homeostasis. Hemichannels are also present in the non-junctional regions of the cell plasma membrane and they provide a direct pathway for communication between the cytoplasm and the extracellular region. Recent studies suggest that gap junctional communication is much more complex than previously anticipated, in terms of both its structure as well as its activity. While the mechanism of gap junction activity is being studied extensively, their quaternary structure, assembly, and conformational changes underlying gating of their activity as well as their physiological role are poorly understood because, due to their complex structure, these junctions are less amenable to existing techniques for high-resolution three-dimensional structure-function analyses. Atomic Force Microscopy (AFM) images molecular structure of biological specimens in an aqueous environment, allows on-line perturbations, and can be coupled with electrophysiological, biochemical, and other microscopic techniques. The present review examines the potential of AFM application for the study of the molecular structure of hydrated, native gap junctions and hemijunctions as well as their physiological functions. Special attention is paid to new, complementary, or provocative findings from AFM studies of both vertebrate and invertebrate gap junctions and hemijunctions.     

Morphological and cytochemical aspects of capillary permeability

Transport of plasma soluble constituents across the capillary wall is of primordial importance in cardiovascular physiology. While physiological experiments have concluded with the existence of two sets of pores, a large one responsible for the transport of proteins and a small one designed for the diffusion of small solutes, the morphological counterparts have yet to get general agreement. In this review, we present the different proposed paths within and between the endothelial cells that do allow passage of plasma constituents and may respond to the definitions established by physiological means. The vesicular system existing in endothelial cells has been the first transendothelial path to be proposed. Several data have demonstrated the involvement of this system in transport, although others have systematically brought controversy. One alternative to the vesicles has been the demonstration of membrane-bound tubules creating, in certain cases, transendothelial channels that would allow diffusion of plasma proteins and other constituents across the capillary wall. Access to this tubulo-vesicular system could be restrained by the stomatal diaphragm and facilitated by specific membrane receptors. Further, we have demonstrated for the first time with morpho-cytochemical tools, that the intercellular clefts are the site of diffusion for small molecules such as peptides having a molecular weight inferior to 3,000. For the fenestrated capillary bed, we have shown that fenestrae are the site through which plasma constituents cross the capillary wall. However, and in spite of the existence of these large open pores, the endothelial cells still display the tubulo-vesicular system involved in transport of large molecules and their intercellular clefts are also the site of diffusion of small molecules. Making consensus on the existence of an intracellular tubulo-vesicular system in non-fenestrated capillaries, responsible for the transport of large molecules by the endothelial cells, and understanding the rational for the fenestrated capillary to have three paths for transport--the fenestrae, the tubulo-vesicular system, and the inter-endothelial clefts--require further investigation.     

The torpedo electrocyte: a model system to study membrane-cytoskeleton interactions at the postsynaptic membrane

Many aspects of the organization of the electromotor synapse of electric fish resemble the nerve-muscle junction. In particular, the postsynaptic membrane in both systems share most of their proteins. As a remarquable source of cholinergic synapses, the Torpedo electrocyte model has served to identify the most important components involved in synaptic transmission such as the nicotinic acetylcholine receptor and the enzyme acetylcholinesterase, as well as proteins associated with the subsynaptic cytoskeleton and the extracellular matrix involved in the assembly of the postsynaptic membrane, namely the 43-kDa protein-rapsyn, the dystrophin/utrophin complex, agrin, and others. This review encompasses some representative experiments that helped to clarify essential aspects of the supramolecular organization and assembly of the postsynaptic apparatus of cholinergic synapses.     

The behaviour of the plasma membrane during plasmolysis: a study by UV microscopy

A high resolution ultraviolet (UV) bright-field microscope was used to analyse the formation of Hechtian strands and the Hechtian reticulation that remain attached to the cell wall after plasmolysis and deplasmolysis of onion inner epidermal cells. In real time video images, UV microscopy allowed a detailed investigation of the dynamic behaviour of the plasma membrane during the processes of osmotic water loss and uptake. Furthermore, the role of cytoskeletal elements as possible linkers of the plasma membrane to the cell wall was probed by application of cytoskeletal drugs during plasmolysis. Microtubules were depolymerized in oryzalin, and latrunculin B was used to destabilize actin microfilaments. The results showed no visible changes in the formation of the Hechtian reticulation or strands. Plasmolysis forms appeared to be normal, indicating stong membrane-to-wall attachments independent of cytoskeletal elements. During re-expansion of the protoplast in deplasmolysis, the plasma membrane incorporated Hechtian strands and subprotoplasts, fused with the Hechtian reticulation and finally realigned at the cell wall.     

Cardiac ischemic preconditioning prevents dystrophin proteolysis by MMP-2 inhibition

Dystrophin is a membrane-associated protein responsible for structural stability of the sarcolemma in cardiac myocytes and is very sensitive to ischemic damage. The goal of our study was to determine if ischemic preconditioning could prevent dystrophin breakdown through inhibition of matrix metalloproteinase-2 (MMP-2) activity. Isolated rabbit hearts were subjected to global ischemia with or without reperfusion in order to evaluate if dystrophin is preserved by ischemic preconditioning through MMP-2 inhibition. Ischemic preconditioning significantly reduced the infarct size induced by 30 min of ischemia and 180 min of reperfusion. Importantly, it also diminished dystrophin proteolysis and attenuated MMP-2 activity after 30 min ischemia. Thus, our study shows a novel protective role of ischemic preconditioning as a mechanism of preservation of plasma membrane integrity by inhibiting MMP-2 activation.     

Time-lapse imaging of In Vitro myogenesis using atomic force microscopy

Myoblast therapy relies on the integration of skeletal muscle stem cells into distinct muscular compartments for the prevention of clinical conditions such as heart failure, or bladder dysfunction. Understanding the fundamentals of myogenesis is hence crucial for the success of these potential medical therapies. In this report, we followed the rearrangement of the surface membrane structure and the actin cytoskeletal organization in C2C12 myoblasts at different stages of myogenesis using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging of living myoblasts undergoing fusion unveiled that within minutes of making cell–cell contact, membrane tubules appear that unite the myoblasts and increase in girth as fusion proceeds. CLSM identified these membrane tubules as built on scaffolds of actin filaments that nucleate at points of contact between fusing myoblasts. In contrast, similarly behaving membrane tubules are absent during cytokinesis. The results from our study in combination with recent findings in literature further expand the understanding of the biochemical and membrane structural rearrangements involved in the two fundamental cellular processes of division and fusion.     

亲和超滤质谱技术在中药活性成分筛选中的研究进展

亲和超滤质谱技术是20世纪90年代中期发展起来的一种快速、简单、有效的药物小分子发现模式。该技术利用配体与受体之间特异性结合,通过超滤装置快速筛选活性小分子化合物,再结合液相色谱 质谱联用技术（LC-MS）,鉴定活性成分结构。亲和超滤质谱技术集药物活性成分筛选、结构鉴定于一体,尤其适用于从复杂体系中筛选潜在的活性小分子化合物。近年来,针对中药发挥药理作用具有多组分、多靶点的重要特点,亲和超滤质谱技术已被广泛用于从中药提取物中筛选与特定蛋白靶点相结合的小分子活性物质,对阐明中药药效的物质基础和以活性成分作为先导化合物的新药开发具有重要意义,是对传统药物发现方法的有利补充。本工作综述了该技术在中药活性成分筛选中的原理、特点、应用进展,以及对未来的展望。     

Cytoskeletal architecture of neuromuscular junction: Localization of vinculin

Cytoskeletons underneath the postsynaptic membrane of neuromuscular junctions were studied by using a quick-freeze deep-etched method and immunoelectron microscopy of ultrathin frozen sections. In a quick-freeze deep-etched replica of fresh, unfixed muscles, 8.9 ± 1.5-nm particles were present on the true postsynaptic membrane surface. Underneath this receptor-rich postsynaptic membrane, networks of fine filaments were observed. These cytoskeletal networks were more clearly observed in extracted samples. In these samples, diameters of the filaments which formed networks were measured. In the platinum replica, three kinds of filament were recognized—12 nm, 9 nm, and 7 nm in diameter. The 12-nm filament seemed to correspond to the intermediate filament. The other two filaments formed meshworks between intermediate filaments and plasma membrane. In ultrathin frozen sections vinculin label was localized just beneath the plasma membrane. Thirty-six percent of the label was within 18 nm from the cytoplasmic side of the plasma membrane and 50% was within 30 nm. Taking the size of the vinculin molecule into account, it was concluded that vinculin is localized just beneath the plasma membrane and might play some role in anchoring filaments which formed meshworks underneath the plasma membrane.     

Dissection of Nod factor signalling in legumes: cell biology, mutants and pharmacological approaches

Nodulation factors (NFs) are lipochito‐oligosaccharide signal molecules excreted by soil‐living rhizobia. These molecules elicit a range of responses in the legume roots, with which the bacteria can live in symbiosis. In this review we focus on the genetic, pharmacological and cell biological approaches that have been, and are being, undertaken to decipher the signalling pathways that lead to the symbiotic responses in the plant.     

Vascular wall: potential target for the physicochemical effects of cis-unsaturated free fatty acids

Diets rich in monounsaturated cis-FFA (cis FFA) are associated with a significant reduction of cardiovascular risk. Although several different mechanisms have been proposed to explain this protective effect, the biochemical processes involved have not been fully elucidated. It has been shown that upon their incorporation into the plasma membrane, cis FFA induce a marked perturbation of the lipid domains, altering membrane fluidity as well as lipid-lipid and lipid-protein interactions in the bilayer plane. During the last few years, several lines of evidence have shown that these perturbations disrupt the activity of several membrane proteins and enzymatic systems. As a result, several critical transmembrane signaling systems, including the Ins(1,4,5)P(3)/DAG/[Ca(2+)](i), the cAMP/PKA, and the voltage-operated Ca(2+) influx are strongly inhibited by cis FFA in different experimental models. Furthermore, this inhibition is associated with alterations in the timing of the cell cycle as well as in the final steps of the secretory pathway. We propose that this complex set of biological actions exerted by cis FFA at the plasma membrane may contribute to explain the protective roles that these molecules appear to exert on the vascular wall.     

Application of WGA lectin staining for visualization of the connective tissue in skeletal muscle, bone, and ligament/tendon studies

During immunostaining of specific proteins in tissue sections using monoclonal and polyclonal antibodies, visualization of general tissue staining/background or major structural features is helpful to pinpoint precise localization of the protein of interest. Often in skeletal muscle research, immunostaining with antibodies against connective tissue or plasma membrane proteins (collagen 1, laminin, and caveolin 3) are used for this purpose. Although immunostaining for these proteins works well, it is time consuming, costly, limits the number of antibodies against protein of interest that can be used on a single section, and is not applicable to some staining techniques. Lectins were frequently used in earlier publications for skeletal muscle fiber boundaries and connective tissue visualization, but are not common in the current research studies. This work investigates costaining of muscle, bone, ligament, and tendon tissue sections with fluorescently tagged wheat germ agglutinin (WGA) lectin as a tool for the visualization of connective tissue. The results of this study show that fluorescent WGA lectin costaining is a cost-effective, fast, and convenient method for connective tissue visualization, especially in the studies where extensive washes reduce staining of the structures that are the primary interest of the investigation.