Integrins as receptors for laminins

Laminins are a family of trimeric glycoproteins present in the extracellular matrix and the major constituents of basement membranes. Integrins are alpha beta transmembrane receptors that play critical roles in both cell-matrix and cell-cell adhesion. Several members of the integrin family, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, alpha 7 beta 1 and alpha 6 beta 4 heterodimers serve as laminin receptors on a variety of cell types. This review summarizes recent advances in understanding the involvement of individual integrins in cell interactions with laminins and the roles of laminin-binding integrins in adhesion-mediated events in vertebrates, including embryonic development, cell migration and tumor cell invasiveness, cell proliferation and differentiation, as well as basement membrane assembly. We discuss the regulation of integrin function via alternative splicing of cytoplasmic domains of alpha and beta subunits of the integrin receptors for laminins and present examples of functional collaboration between laminin-binding integrins and non-integrin laminin receptors. Advances in our understanding of the laminin-binding integrins continue to demonstrate the essential roles these receptors play in maintaining cell polarity and tissue architecture.     

Laminins and human disease

The laminin protein family has diverse tissue expression patterns and is involved in the pathology of a number of organs, including skin, muscle, and nerve. In the skin, laminins 5 and 6 contribute to dermal-epidermal cohesion, and mutations in the constituent chains result in the blistering phenotype observed in patients with junctional epidermolysis bullosa (JEB). Allelic heterogeneity is observed in patients with JEB: mutations that results in premature stop codons produce a more severe phenotype than do missense mutations. Gene therapy approaches are currently being studied in the treatment of this disease. A blistering phenotype is also observed in patients with acquired cicatricial pemphigoid (CP). Autoantibodies targeted against laminins 5 and 6 destabilize epithelial adhesion and are pathogenic. In muscle cells, laminin alpha 2 is a component of the bridge that links the actin cytoskeleton to the extracellular matrix. In patients with laminin alpha 2 mutations, the bridge is disrupted and mature muscle cells apoptose. Congenital muscular dystrophy (CMD) results. The role of laminin in diseases of the nervous system is less well defined, but the extracellular protein has been shown to serve an important role in peripheral nerve regeneration. The adhesive molecule influences neurite outgrowth, neural differentiation, and synapse formation. The broad spatial distribution of laminin gene products suggests that laminin may be involved in a number of diseases for which pathogenic mechanisms are still being unraveled.     

Freeze-Fracture of physiologically identified neuromuscular junctions from single frog muscle fibers

We developed a technique for freeze-fracturing single physiologically identified neuromuscular junctions. This technique permits direct comparison of quantal content with morphological variables such as active zone length per unit terminal length for the same cell. The technique was developed to elucidate the structural basis for variability in transmitter release at the neuromuscular junction. The procedures were as follows: (1) record quantal content by conventional intracellular recordings; (2) mark cells for identification by fluorescent dye injection; (3) fix and stain endplate cholinesterase; (4) glycerinate and remove single fibers from the muscle; (5) draw endplate morphology; (6) freeze-fracture single muscle fibers; (7) examine in a transmission electron microscope; and (8) photograph and measure nerve terminal membrane ultrastructure. We found that approximately 15% of freeze-fractured single muscle fibers exhibited nerve terminal active zones. To demonstrate the usefulness of this technique, physiological and morphological information from an identified junction is presented. Freeze-fracture of identified cells has several advantages over thin sections, which cannot accurately show such things as active zone length, spacing, or intramembrane particles. This technique also has applications to the study of active zone ultrastructure in situations where neurotransmitter release is known to differ from normal levels. In addition, direct correlations between membrane structure and function can be studied in other preparations by this method.     

Ultrastructure of motor nerve terminals in the anterior third of wistar rat tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957 . For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4°C for 2 h, according to the technique described by Tanaka, 1989 . Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi's apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Merosin and congenital muscular dystrophy

Merosin (also called as Laminin-2) is an isoform of laminin comprised of the alpha2, beta1 and gamma1 chains. In European populations, half of the patients with classical congenital muscular dystrophy have mutations of the LAMA2 gene (6q22-23) and present reduced or absence of laminin alpha2 chain. This form is generally referred to as merosin-deficient CMD. Merosin-deficient CMD is characterized by involvement of not only skeletal muscle but also central and peripheral nervous systems: Extensive brain white matter abnormalities are found by magnetic resonance imaging (MRI). However, most patients show no mental retardation. Recent case studies reported that some patients have several structural abnormalities such as abnormal cerebral cortical gyration, hypoplasia of cerebellum and pons, and dilation of ventricles. At present, functions of merosin related to muscle degeneration have not been fully elucidated. In addition, the mechanisms responsible for pathogenesis of diffuse brain white matter abnormalities remain to be determined. As mouse models for merosin-deficient CMD, three spontaneous mutants(dy, dy(2J), dy(PAS1)) and two mutants named dy(W) and dy(3K) by targeted gene disruption have been reported. These mice will help to elucidate the pathogenesis of merosin-deficient CMD and serve to develop therapy.     

The ultrastructure of neuronal-pinealocytic interconnections in the monkey pineal.

Recent ultrastructural studies of neuronal-pinealocytic interconnections in the monkey pineal are reviewed. The pinealocytes in the adult monkey show almost all of the cytological specializations known in subprimate mammals. Adjacent pinealocytes are functionally coupled through ribbon synapses on cell bodies and gap junctions on cell bodies and cell processes. The pinealocytes receive direct synpatic contacts of nerve fibers with cholinergic terminal morphology. Nerve cells restricted to the central portion of the pineal receive synaptic contacts with more than three different morphologically defined types of nerve terminals. In addition to nerve terminals containing small clear vesicles or vesicles of pleomorphic morphology, a pinealocyte's terminal process containing the synaptic ribbon forms a true synaptic contact on the nerve cell body. The diversity of synapses on these nerve cells strongly suggests multiple origins of these neurons rather than a single peripheral parasympathetic origin. The possible involvement of pineal neurons in an intrinsic circuit that regulates the function of pinealocytes and integrates the neural input from the central as well as the peripheral nervous systems is discussed.     

Drosophila larval neuromuscular junction: molecular components and mechanisms underlying synaptic plasticity

Understanding the mechanisms that mediate synaptic plasticity is a primary goal of molecular neuroscience. The Drosophila larval neuromuscular junction provides a particularly useful model for investigating the roles of synaptic components in both structural and functional plasticity. The powerful molecular genetics of this system makes it possible to uncover new synaptic components and signaling molecules, as well as their function in the intact organism. Together with the mouse hippocampus and Aplysia dissociated cell culture, the Drosophila larval neuromuscular junction has been among the most valuable model systems for examining the molecular and cellular basis of neuronal plasticity.     

Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix

Angiogenesis is critical to wound repair. Newly formed blood vessels participate in provisional granulation tissue formation and provide nutrition and oxygen to growing tissues. In addition, inflammatory cells require the interaction with and transmigration through the endothelial basement membrane to enter the site of injury. Angiogenesis, in response to tissue injury, is a dynamic process that is highly regulated by signals from both serum and the surrounding extracellular matrix (ECM) environment. Vascular endothelial growth factor, angiopoietin, fibroblast growth factor, and transforming growth factor beta are among those most potent angiogenic cytokines in wound angiogenesis. The cooperative regulation of them is essential for wound repair. Migration of endothelial cells and development of new capillary vessels during wound repair is dependent on not only the cells and cytokines present but also the production and organization of ECM components both in granulation tissue and in endothelial basement membrane. The ECM regulates angiogenesis by providing scaffold support and signaling roles. They also serve as a reservoir and modulator for growth factors. Laminins are the major noncollagenous ECM of endothelial basement membrane. Two newly recognized laminins, 8 and 10, are the major laminins produced by human dermal microvascular endothelial cells. Laminin 10 is highly expressed in blood vessels around skin wounds. Laminin 8 promotes dermal endothelial cell attachment, migration, and tubule formation. Integrins with either beta 1 or alpha v subunits are the major cellular surface receptors for ECM molecules and mediate the interactions between cells and ECM during wound angiogenesis.     

Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: refinements and applications

Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve-muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better-defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon-induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon-induced AChR aggregation on muscle cell species, by the use of chick-rat chimeric co-cultures. (5) We provide evidence for the role of alternatively-spliced agrin isoforms in synapse formation by using single cell RT-PCR with neurons collected from co-cultures after observation of axon-induced AChR aggregation. Microsc. Res. Tech. 49:26-37, 2000. Published 2000 Wiley-Liss, Inc.     

Myotonic dystrophy protein kinase of the cardiac muscle: evaluation using an immunochemical approach

Myotonic dystrophy (DM) is an inherited multisystem disorder characterized by the presence of a high polymorphic expansion of trinucleotide (CTG) repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. However, the role of myotonic dystrophy protein kinase (DMPK) has yet to be elucidated. Studies aimed to discover possible physiological targets of DMPK indicated several subcellular localization sites, such as neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum in the skeletal muscle and intercalated discs in the cardiac muscle. Here, we extend our previous observations on the localization of DMPK at gap junction (GJ) level in the heart, taking advantage of the polyclonal peptide-specific anti-DMPK antibodies raised against two different domains of the protein. DMPK was detected by immunofluorescence at the intercalated disc level by both antibodies. Double immunofluorescence staining experiments performed with each anti-DMPK and anti-connexin43 showed colocalization of the two antigens. Immunoblot analysis of partially purified GJs showed co-sedimentation of DMPK and connexin43. We conclude that GJs are a genuine localization site of DMPK. Given the known regulation exerted by protein kinases on assembly, trafficking, gating, and disassembly of connexins, such a localization may be relevant to the functional role of connexins. DM is the most common muscular dystrophy in adults, and is known by the cardiac involvement that is a common feature in DM patients. Localization of DMPK at GJ in relation to DM is also briefly discussed.     

S-100 proteins in the human peripheral nervous system

This article reviews the distribution of S100 proteins in the human peripheral nervous system. The expression of S100 by peripheral glial cells seems to be a distinctive fact of these cells, independently of their localization and their ability to myelinate or not. S100 proteins expressing cells include satellite cells of sensory, sympathetic and enteric ganglia, supporting cells of the adrenal medulla, myelinating and non-myelinating Schwann cells in the nerve trunks, and the Schwann-related cells of sensory corpuscles. In addition, S100 proteins are expressed in peripheral neurons. Most of them express S100alpha protein, and a subpopulation of sensory neurons in dorsal root ganglia contains S100beta protein or S100alpha plus S100beta proteins.     

Ultrastructural aspects of mouse nerve‐muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX‐I and Bbil‐TX: Unknown myotoxic effects

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve‐muscle preparations exposed to Bothrops venoms and their phospholipase A₂ toxins (PLA₂) can exhibit a neurotoxic pattern as increase in frequency of miniature end‐plate potentials (MEPPs) as well as in amplitude of end‐plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA₂ toxins (BthTX‐I and Bbil‐TX) in mouse isolated nerve‐phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX‐I and Bbil‐TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic‐like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.     

Role of nitric oxide and nitric oxide synthases in experimental models of denervation and reinnervation

Nitric oxide (NO) is a short-living free molecule synthesized by three different isoforms of nitric oxide synthases (NOS)—neuronal NOS, endothelial NOS, and inducible NOS—associated with neuromuscular transmission, muscle contractility, mitochondrial respiration, and carbohydrate metabolism in skeletal muscle. Neuronal NOS is constitutively expressed at the muscle fiber sarcolemma linked to the dystrophin-glycoprotein complex and concentrated at the neuromuscular endplate. There is increasing evidence that altered expression of neuronal NOS plays a role in muscle fiber damage in neuromuscular diseases such as dystrophinopathies and denervating disorders. Although there have been some previous conflicting results on the neuronal NOS expression pattern in denervated muscle fibers, it is now well established that denervation is associated with a down-regulation and disappearance of sarcolemmal neuronal NOS at synaptic/extrasynaptic or both sites. As NO has been shown to induce collapse and growth arrest on neuronal growth cones, down-regulation of sarcolemmal neuronal NOS may contribute to axonal regeneration and attraction to muscle fibers aiming at the formation of new motor endplates providing reinnervation and reconstitution of NOS expression. As NO serves as a retrograde messenger, it may trigger structural downstream events responsible for neuromuscular synaptogenesis and preventing polyneural innervation. Nevertheless, decreased NO production in denervation reduces the cytoprotective scavenger function of NO for superoxide anions promoting oxidative stress that is likely to be involved in muscle fiber damage and death. However, the multifaced role of NOS and NO under physiological and pathological conditions remains poorly understood on the basis of the current knowledge. Microsc. Res. Tech. 55:181–186, 2001. © 2001 Wiley-Liss, Inc.     

Regeneration of periodontal Ruffini endings in adults and neonates

We reviewed the regeneration of periodontal Ruffini endings, primary mechanoreceptors in the periodontal ligament, following injury to the inferior alveolar nerve (IAN) in adult and neonatal rats. Morphologically, mature Ruffini endings are characterized by an extensive arborization of axonal terminals and association with specialized Schwann cells, called lamellar or terminal Schwann cells. Following injury to IAN in the adult, the periodontal Ruffini endings of the rat lower incisor ligament regenerate more rapidly than Ruffini endings in other tissues. During regeneration, terminal Schwann cells migrate into regions where they are never found under normal conditions. The development of periodontal Ruffini endings of the rat incisor is closely associated with the eruption of the teeth; the morphology and distribution of the terminal Schwann cells became almost identical to those in adults during postnatal days 15-18 (PN 15-18d) when the first molars appear in the oral cavity, while the axonal elements showed extensive ramification around PN 28d when the functional occlusion commences. When the IAN was injured in neonates, the regeneration of periodontal Ruffini endings was delayed compared with the adults. The migration of terminal Schwann cells is also observed following IAN injury, after which the distribution of terminal Schwann cells became almost identical to that of the adults, i.e., PN 14d. Since the interaction between axon and Schwann cell is important during regeneration and development, further studies are required to elucidate its molecular mechanism during the regeneration as well as the development of the periodontal Ruffini endings.     

Immunoelectron microscopic studies on protein gene product 9.5 and calcitonin gene-related peptide in vallate taste cells and related nerves in the guinea pig

On the basis of our previous report that protein gene product 9.5 (PGP 9.5)-immunoreactive nerve fibers and taste cells and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers are found in guinea pig vallate papillae [Huang and Lu (1996b) Arch. Histol. Cytol. 59:433-441]. We speculated that PGP 9.5 might be a marker for taste receptor cells and that CGRP might play an important role in taste transmission. We, therefore, performed an immunohistochemical and ultrastructural analysis of taste cells and related nerves in guinea pig vallate papillae. In the connective tissue of the vallate papilla, the ultrastructural data revealed that the PGP 9.5-immunoreactive nerve fibers were both myelinated and unmyelinated. The CGRP-immunoreactive nerve fibers were unmyelinated and surrounded by the cytoplasm of Schwann cells as were the non-immunoreactive fibers. In the vallate taste buds, only type III cells, which make synaptic contacts with intragemmal nerves, were PGP 9.5-immunoreactive, while the nerve terminals making synaptic contact with the underlying type III cells were CGRP-immunoreactive. From these observations, we conclude that: (1) PGP 9.5 might be a useful specific marker for type III cells in guinea pig vallate taste buds; and (2) CGRP-containing nerve fibers might be primarily involved in the neural transmission of taste stimuli.     

Clustering of sodium channels at the neuromuscular junction

Voltage-gated sodium channels (NaChs) are highly concentrated in the postsynaptic region of the neuromuscular junction, especially in the depths of postsynaptic folds and in the perijunctional region. The formation of the high NaCh density occurs during synapse maturation, approximately 2 weeks after initial synaptic contact in the rodent. The concentration of NaChs and their localization in the troughs of the folds increase the safety factor for neuromuscular transmission by reducing the threshold for initiation of the action potential. There is evidence that agrin plays a role in the formation of NaCh aggregation. Molecules such as ankyrin and syntrophin that bind NaChs may be important for maintenance of the high channel density at the endplate.     

Integrins as mediators of epithelial cell-matrix interactions in the human small intestinal mucosa

The intestinal epithelium is a highly dynamic tissue, which depends on a variety of factors for the regulation of its rapid renewal and expression of digestive functions. Over the last 10 years, it has become evident that among these factors are cell interactions with the extracellular matrix, more specifically with the underlying basement membrane, through a series of specific cell membrane receptors, many of which are integrins. Integrins regulate the assembly of adhesive junctions as well as the activation of various signaling pathways, leading to the modulation of gene expression. The analysis of the integrin repertoire along the crypt-villus axis in the human small intestinal epithelium identifies a number of beta1 and beta4 integrins, showing differential patterns of expression relative to its two functional compartments. Among them are the integrins alpha3beta1, alpha7Bbeta1 and the functional form of alpha6beta4 that appear to be related, in concert with the distribution of their ligands, to the process of intestinal cell differentiation, and the integrins alpha2beta1, alpha1beta1, alpha5beta1, and the non-functional form of alpha6beta4 that seem to be coupled with the undifferentiated/proliferative status of crypt cells. These observations delineate the potential complexity of the organization of epithelial cell-matrix interactions involved in the maintenance of the human intestinal crypt-villus axis.     

Inactivation of myosin heavy chain genes in the mouse: diverse and unexpected phenotypes

Myosin heavy chain (MyHC) is a critical component of the cellular contractile apparatus. The mammalian genome contains two nonmuscle, two smooth muscle, and eight striated muscle isoforms of MyHC. Within each class of genes, there is extremely high sequence homology among different MyHC isoforms, raising the question of whether these isoforms are functionally redundant or whether they perform unique roles in cell function. Recently, strains of mice null for four different MyHC isoforms have been generated. Mice null for the nonmuscle II-B isoform experience significant prenatal lethality and surviving animals have several cardiac abnormalities [Tullio et al. (1997) Proc Natl Acad Sci USA 94:12407-12412]. Mice homozygous null for alpha cardiac MyHC are embryonic lethal, while heterozygous mice are viable but also have numerous cardiac defects [Jones et al. (1996) J Clin Invest 98:1906-1917]. Mice null for IIb or IId adult skeletal MyHC are viable but have skeletal muscle abnormalities compared to wild type mice, despite compensation of a neighboring MyHC gene [Acakpo-Satchivi et al. (1997) J Cell Biol 139:1219-1229]. Both IIb and IId null mice show significant decreases in body mass. Mean muscle mass is also significantly decreased in both null strains but the extent and the pattern of affected muscles differs between the two strains. Both strains show evidence of skeletal muscle pathology but again the pattern and extent differ between the two strains. Finally, both adult skeletal strains demonstrate distinct impairments in contractile function when compared to wild type. Together these observations support the hypothesis that the different isoforms of MyHC are functionally unique and cannot substitute for one another.