The Basement Membrane/Basal Lamina of Skeletal Muscle
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.r200027200
ISSN1083-351X
Autores Tópico(s)Muscle Physiology and Disorders
Resumobasement membrane basal lamina acetylcholinesterase acetylcholine receptor muscle-specific kinase Many cells, including skeletal muscle fibers, are coated by a layer of extracellular matrix material called the basement membrane (BM).1 The BM, in turn, is composed of two layers: an internal, felt-like basal lamina (BL) directly linked to the plasma membrane, and an external, fibrillar reticular lamina. BMs contain protein and carbohydrate but no lipid or nucleic acid. Virtually all the protein is glycosylated, and nearly all the carbohydrate is covalently bound to protein. The fibrils of the reticular lamina are collagenous, and they are embedded in an amorphous proteoglycan-rich ground substance. The BL contains non-fibrillar collagen, non-collagenous glycoproteins, and proteoglycans (1Timpl, R., and Rohrbach, O. (eds) Molecular and Cellular Aspects of Basement Membranes, Academic Press, New York.Google Scholar). Initially, the BM was viewed as a static structure that provides mechanical support; essentially something for the cells to sit on. A key advance was the discovery that, because the acellular BM survives injury to associated cells, it can provide a scaffold to orient and constrain cells during regeneration (2Vracko R. Benditt E.P. J. Cell Biol. 1972; 55: 406-419Crossref PubMed Scopus (276) Google Scholar). A more radical transformation over the past few decades was the realization that BM components play active roles and that these roles extend to developmental as well as regenerative processes (1Timpl, R., and Rohrbach, O. (eds) Molecular and Cellular Aspects of Basement Membranes, Academic Press, New York.Google Scholar). In skeletal muscle, these processes include myogenesis and synaptogenesis. Most recently, emphasis has shifted to a search for the matrix-associated signals and membrane-associated receptors that underlie cell-matrix interactions. The purpose of this minireview is to relate results from the new molecular analyses to the early cellular observations that motivated them. For more detailed descriptions of what happened in between, see Refs. 3Sanes J.R. Engel A.G. Franzini-Armstrong C. Mycology. McGraw Hill, New York1994: 242-260Google Scholar, 4Sanes J.R. Semin. Dev. Biol. 1995; 6: 163-173Crossref Scopus (28) Google Scholar, 5Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1209) Google Scholar. Although we now know that BMs are present in nearly all tissues, their existence was first appreciated in muscle. In his 1840 report "On the Minute Structure and Movements of Voluntary Muscle," Bowman (6Bowman W. Philos. Trans. R. Soc. Lond. Biol. Sci. 1840; 130: 457-494Crossref Google Scholar) described a "highly delicate, transparent, and probably elastic" sheath encircling individual muscle fibers. This sheath, which he called the sarcolemma, became apparent when muscle fibers were injured during dissection; the cell itself lysed and retracted, leaving the sarcolemma behind (Fig.1). Over a century later, electron microscopy revealed that the BM is the main component of such tubes and that the BL is a main component of the BM. Today the term sarcolemma is often used to refer to the plasma membrane alone, although only fragments of it were present in Bowman's tubes (3Sanes J.R. Engel A.G. Franzini-Armstrong C. Mycology. McGraw Hill, New York1994: 242-260Google Scholar). The terms BL and BM are often used interchangeably, but should not be; I will attempt to use them appropriately here. Bowman's view of what we now know to be BM and particularly his evidence for its "strength and tenacity" (6Bowman W. Philos. Trans. R. Soc. Lond. Biol. Sci. 1840; 130: 457-494Crossref Google Scholar) led directly to appreciation of its role in muscle function. Muscles are strong, flexible, and stress-resistant. Formal models of their mechanical properties include both contractile and elastic elements. The contractile element is, of course, the sarcomere, and extracellular matrix accounts for much of the elasticity. In fact, several matrix-rich structures contribute to muscle strength and elasticity, but a sizable fraction has been shown to reside in the BM (7Tidball J.G. Biophys. J. 1986; 50: 1127-1138Abstract Full Text PDF PubMed Scopus (35) Google Scholar). Direct biophysical analysis of BM is lacking, but keys to its strength most likely are its major structural components (8Timpl R. Brown J.C. Bioessays. 1996; 18: 123-132Crossref PubMed Scopus (575) Google Scholar, 9Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1024) Google Scholar). The most abundant protein of the BL is triple-helical collagen IV, the subunits of which, called α chains, have prominent terminal non-collagenous domains. The major non-collagenous protein is laminin, which is also a heterotrimer of related chains, in this case called α, β, and γ. Both collagens IV and laminins exist in multiple isoforms, with the most abundant in muscle being collagen (α1(IV))2(α2(IV))1 and laminin α2β1γ1 (also called laminin-2). The basic structure of BLs appears to involve distinct networks of collagens IV and laminin, each of which is capable of self-assembly. The collagen network becomes cemented by covalent cross-links, and the two networks are linked to each other by another non-collagenous glycoprotein, entactin/nidogen. These core components bear a multitude of recognition sites that bind other BL components, anchor reticular lamina components to the BL, and serve as ligands for membrane-associated receptors. Among the transmembrane receptors are the integrins and dystroglycans, both of which interact with the cytoskeleton (10Michele D.E. Campbell K.P. J. Biol. Chem. 2003; 278 (January 29, 10.1074/jbc.R200031200)Abstract Full Text Full Text PDF Scopus (366) Google Scholar, 11Mayer U.R. J. Biol. Chem. 2003; 278: 14587-14590Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Thus, one can envision a series of direct linkages that together span the distance from reticular lamina to BL to plasma membrane to cytoskeleton. The BM provides a significant fraction of the tensile strength of the whole structure (3Sanes J.R. Engel A.G. Franzini-Armstrong C. Mycology. McGraw Hill, New York1994: 242-260Google Scholar), presumably via the collagen/laminin networks of BL, which run orthogonal to this axis. Genetic studies of muscle disease show that the BM is critical for the maintenance of muscle integrity. Positional cloning in humans and analysis of naturally occurring and targeted mutants in mice have revealed that muscular dystrophy can arise from loss of any of several components in the reticular lamina-BL-membrane-cytoskeleton linkage. These include laminin α2 (congenital muscular dystrophy), its major transemembrane receptors, integrin α7 and dystroglycan; dystrophin, which links dystroglycan to the cytoskeleton (Duchenne muscular dystrophy); the dystroglycan- and dystrophin-associated sarcoglycans (limb-girdle muscular dystrophies); and the α chains of collagen VI, which help connect the BL to the reticular lamina (Bethlem myopathy) (10Michele D.E. Campbell K.P. J. Biol. Chem. 2003; 278 (January 29, 10.1074/jbc.R200031200)Abstract Full Text Full Text PDF Scopus (366) Google Scholar, 11Mayer U.R. J. Biol. Chem. 2003; 278: 14587-14590Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 12Helbling-Leclerc A. Zhang X. Topaloglu H. Cruaud C. Tesson F. Weissenbach J. Tome F.M. Schwartz K. Fardeau M. Tryggvason K. et al.Nat. Genet. 1995; 11: 216-218Crossref PubMed Scopus (558) Google Scholar, 13Xu H. Wu X.R. Wewer U.M. Engvall E. Nat. Genet. 1994; 8: 297-302Crossref PubMed Scopus (320) Google Scholar, 14Blake D.J. Weir A. Newey S.E. Davies K.E. Physiol. Rev. 2002; 82: 291-329Crossref PubMed Scopus (886) Google Scholar, 15Jobsis G.J. Keizers H. Vreijling J.P. de Visser M. Speer M.C. Wolterman R.A. Baas F. Bolhuis P.A. Nat. Genet. 1996; 14: 113-115Crossref PubMed Scopus (215) Google Scholar). Importantly, in all of these diseases, muscles develop normally but then degenerate. Thus, even though the BL does play roles in myogenesis (see below) it is separately required for muscle maintenance. In part, this requirement may be a passive, mechanical one, but more active mechanisms also contribute. The core BL components, laminin and collagen IV, are signaling as well as structural molecules, and their receptors, dystroglycan and integrins, are signal transducers. For example, active signaling from laminin α2 may provide a survival signal for muscle, and its absence in congenital dystrophy is associated with particularly high levels of apoptosis (16Vachon P.H. Loechel F. Xu H. Wewer U.M. Engvall E. J. Cell Biol. 1996; 134: 1483-1497Crossref PubMed Scopus (193) Google Scholar). In short, muscle maintenance requires both the structural and signaling properties of BL. In one of the first clear demonstrations that extracellular matrix influences cellular differentiation, Hauschka and Konigsberg (17Hauschka S.D. Konigsberg I.R. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 119-126Crossref PubMed Scopus (332) Google Scholar) showed that substrate-bound collagen could replace "conditioned medium" factors in promoting the formation of myotubes from cultured myoblasts. Subsequent work showed that several matrix components affect myogenesis. Of these, laminin appears to be particularly critical. Laminin enhances proliferation of myoblasts, stimulates their motility, and leads them to assume the bipolar shape characteristic of fusing cells (18Foster R.F. Thompson J.M. Kaufman S.J. Dev. Biol. 1987; 122: 11-20Crossref PubMed Scopus (133) Google Scholar). Myotube formation is decreased, although not abolished, in the absence of laminin (19Smyth N. Vatansever H.S. Murray P. Meyer M. Frie C. Paulsson M. Edgar D. J. Cell Biol. 1999; 144: 151-160Crossref PubMed Scopus (408) Google Scholar). In contrast, fibronectin selectively promotes adhesion of fibroblasts and may lead to dedifferentiation of myoblasts (20von der Mark K. Ocalan M. Differentiation. 1989; 40: 150-157Crossref PubMed Scopus (108) Google Scholar). The locations of these proteins also differ; laminin adjoins myotubes whereas fibronectin is initially excluded from myogenic regions (20von der Mark K. Ocalan M. Differentiation. 1989; 40: 150-157Crossref PubMed Scopus (108) Google Scholar). Therefore, laminin and fibronectin may be involved in sorting myoblasts from fibroblasts as well as in orchestrating their differentiation. In addition, laminin and collagen IV provide binding sites for proteoglycans, the principal one in muscle being perlecan (8Timpl R. Brown J.C. Bioessays. 1996; 18: 123-132Crossref PubMed Scopus (575) Google Scholar). The glycosaminoglycan chains of the proteoglycans, in turn, provide binding sites that concentrate and present bioactive polypeptides such as fibroblast growth factors and transforming growth factors, which are critical for myogenesis (21Pirskanen A. Kiefer J.C. Hauschka S.D. Dev. Biol. 2000; 224: 189-203Crossref PubMed Scopus (57) Google Scholar, 22Baeg G.H. Perrimon N. Curr. Opin. Cell Biol. 2000; 12: 575-580Crossref PubMed Scopus (94) Google Scholar). Indeed, these nominally soluble factors are predominantly matrix-associated in vivo. Thus, major BL components not only promote myogenesis directly but also orchestrate muscle development by presentation of morphogenic, mitogenic, and trophic factors. Bowman's discovery of the BM arose from its persistence following injury during dissection. When injury occurs in vivo, new muscle fibers regenerate from a resident population of stem cells, called satellite cells, which are wedged between muscle fiber and BL. Bowman (6Bowman W. Philos. Trans. R. Soc. Lond. Biol. Sci. 1840; 130: 457-494Crossref Google Scholar) noted that the BM "provides an effectual barrier between the parts within and those without"; as predicted from this property, most satellite cells remain within the BL as they divide and form myotubes (2Vracko R. Benditt E.P. J. Cell Biol. 1972; 55: 406-419Crossref PubMed Scopus (276) Google Scholar, 23Sanes J.R. Marshall L.M. McMahan U.J. J. Cell Biol. 1978; 78: 176-198Crossref PubMed Scopus (397) Google Scholar). Thus, by constraining the growth and migration of activated satellite cells, BL orients the regeneration of new muscle fibers. From what we know about myogenesis, it seems likely that the BL also actively promotes regeneration. In addition, BL acts as a mechanical barrier to prevent migratory loss of satellite cells from normal muscle and could be involved in repressing satellite cell mitosis and differentiation in the absence of damage. The guidance that BL provides is of functional importance. Muscles do regenerate if the BL is disrupted, but myotubes are not oriented in parallel so the regenerate as a whole may develop little net force (3Sanes J.R. Engel A.G. Franzini-Armstrong C. Mycology. McGraw Hill, New York1994: 242-260Google Scholar). Furthermore, because BLs of nerves and blood vessels also act as scaffolds for regeneration (2Vracko R. Benditt E.P. J. Cell Biol. 1972; 55: 406-419Crossref PubMed Scopus (276) Google Scholar, 24Nguyen Q.T. Sanes J.R. Lichtman J.W. Nat. Neurosci. 2002; 5: 861-867Crossref PubMed Scopus (242) Google Scholar), the integrity of connective tissue favors rapid revascularization and reinnervation. In general, recovery of function is good following injuries that minimally disrupt the integrity and orientation of the sheaths and poor following injuries that destroy these scaffolds. The extracellular matrix is structurally and functionally specialized in areas where muscle abuts tendon or nerve. At the neuromuscular junction, BL but not reticular lamina passes between nerve and muscle membranes and extends into junctional folds that invaginate the postsynaptic membrane (Fig.2). The BL thus constitutes a sizable fraction of the synaptic cleft material of the neuromuscular junction. The cleft is 50-nm-wide, which is a greater distance than that spanned by membrane-associated adhesion molecules (e.g. cadherins). Based on these considerations alone, it is evident that the BL must contribute to the tight adhesion of pre- and postsynaptic partners. Indeed, when muscles are treated with proteases that digest BL but not plasma membrane, nerve terminals lose their firm attachment to the end plate and can easily be pulled away (25Betz W. Sakmann B. J. Physiol. (Lond.). 1973; 230: 673-688Crossref Scopus (118) Google Scholar). Moreover, when muscle is damaged but not denervated, nerve terminals remain at their original sites on the BL for months after the muscle fiber has degenerated (22Baeg G.H. Perrimon N. Curr. Opin. Cell Biol. 2000; 12: 575-580Crossref PubMed Scopus (94) Google Scholar,26Dunaevsky A. Connor E.A. Dev. Biol. 1998; 194: 61-71Crossref PubMed Scopus (17) Google Scholar). Adhesion is likely to be mediated in part by integrins and dystroglycan (27Cohen M.W. Hoffstrom B.G. DeSimone D.W. J. Neurosci. 2000; 20: 4912-4921Crossref PubMed Google Scholar, 28Martin P.T. Kaufman S.J. Kramer R.H. Sanes J.R. Dev. Biol. 1996; 174: 125-139Crossref PubMed Scopus (152) Google Scholar). Other potential adhesive systems are mentioned below. At the myotendinous junction, the surface of the muscle fiber is thrown into invaginations that resemble junctional folds but are deeper. BL extends into these invaginations and is attached to the plasma membrane by periodically arrayed microfibrils (29Benjamin M. Ralphs J.R. Int. Rev. Cytol. 2000; 196: 85-130Crossref PubMed Google Scholar). These fibrils and the increased area of membrane-matrix apposition provided by the invaginations are adaptations for the transmission of force from muscle to tendon. Some molecular differences have been noted between the BL at the myotendinous junction and that coating adjoining regions of the sarcolemma (30Patton B.L. Microsc. Res. Tech. 2000; 51: 247-261Crossref PubMed Scopus (80) Google Scholar, 31Pedrosa-Domellof F. Tiger C.F. Virtanen I. Thornell L.E. Gullberg D. J. Histochem. Cytochem. 2000; 48: 201-210Crossref PubMed Scopus (23) Google Scholar), but the functional significance of these differences is unknown. The key events in neuromuscular transmission are release of acetylcholine from the nerve terminal and activation of acetylcholine receptors in the postsynaptic membrane. One might imagine that the BL would block movement of acetylcholine across the synaptic cleft, but kinetic studies show that its diffusion to receptors is unimpeded (32Land B.R. Harris W.V. Salpeter E.E. Salpeter M.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1594-1598Crossref PubMed Google Scholar). This result is consistent with conclusions reached from analysis of glomerular BL in kidney, which is an effective filter only for macromolecules (33Tryggvason K. Wartiovaara J. Curr. Opin. Nephrol. Hypertens. 2001; 10: 543-549Crossref PubMed Scopus (197) Google Scholar). Thus, diffusion of transmitter to receptors and the passive components of its subsequent dispersal are not significantly affected by BL. On the other hand, the BL is involved in the hydrolysis of acetylcholine by acetylcholinesterase (AChE), which terminates transmitter action faster than would occur by diffusion alone. It was initially believed that AChE was attached to the membrane, as is the case in neuron-neuron synapses. Subsequent studies showed, however, that a major fraction of AChE at the neuromuscular synapse is stably associated with synaptic BL (34Hall Z.W. Kelly R.B. Nat. New Biol. 1971; 232: 62-63Crossref PubMed Scopus (155) Google Scholar, 35McMahan U.J. Sanes J.R. Marshall L.M. Nature. 1978; 271: 172-174Crossref PubMed Scopus (278) Google Scholar). The key to the association is a collagen-like "tail" that is disulfide-bonded to tetramers of catalytic AChE subunits; much of the synaptic enzyme in muscle but little in brain is associated with the tail (35McMahan U.J. Sanes J.R. Marshall L.M. Nature. 1978; 271: 172-174Crossref PubMed Scopus (278) Google Scholar). The tail eluded molecular analysis until recently, but its gene, named ColQ("queue" is French for "tail") has now been cloned and characterized (36Massoulie J. Neurosignals. 2002; 11: 130-143Crossref PubMed Scopus (239) Google Scholar, 37Krejci E. Thomine S. Boschetti N. Legay C. Sketelj J. Massoulie J. J. Biol. Chem. 1997; 272: 22840-22847Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Mutation of the ColQ gene in mice leads to loss of synaptic AChE, and mutations of ColQ in humans underlie some cases of congenital myasthenia gravis (38Feng G. Krejci E. Molgo J. Cunningham J.M. Massoulie J. Sanes J.R. J. Cell Biol. 1999; 144: 1349-1360Crossref PubMed Scopus (147) Google Scholar, 39Shapira Y.A. Sadeh M.E. Bergtraum M.P. Tsujino A. Ohno K. Shen X.M. Brengman J. Edwardson S. Matoth I. Engel A.G. Neurology. 2002; 58: 603-609Crossref PubMed Scopus (43) Google Scholar). ColQ, in turn, binds to perlecan in the BL (40Peng H.B. Xie H. Rossi S.G. Rotundo R.L. J. Cell Biol. 1999; 145: 911-921Crossref PubMed Scopus (183) Google Scholar, 41Arikawa-Hirasawa E. Rossi S.G. Rotundo R.L. Yamada Y. Nat. Neurosci. 2002; 5: 119-123Crossref PubMed Scopus (142) Google Scholar). It is a fascinating testament to the adaptive powers of the synapse that genetic loss of ColQ or AChE is detrimental but not fatal, whereas acute inactivation of AChE by nerve gas leads to fatal respiratory paralysis. Following peripheral nerve injury, motor axons regenerate to form new neuromuscular junctions. Over 100 years ago, Tello (42Ramon y Cajal S. Degeneration and Regeneration of the Nervous System. Oxford Press, New York1928: 265-280Google Scholar) reported that the regenerating axons show a remarkable preference for original synaptic sites. Indeed, when trauma to nerve and muscle are minimized, over 95% of the contacts formed by regenerating axons on muscle fibers occur at original sites, even though these sites occupy only about 0.1% of the muscle fiber surface (5Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1209) Google Scholar). Some of this precision reflects regrowth of axons along the connective tissue pathways that had been associated with the original nerve, a process in which the nerve BL plays a prominent role (24Nguyen Q.T. Sanes J.R. Lichtman J.W. Nat. Neurosci. 2002; 5: 861-867Crossref PubMed Scopus (242) Google Scholar). Once the axons reach denervated muscle fibers, however, they reoccupy original sites at a submicron level of precision, demonstrating the existence of recognition factors closely associated with the muscle fiber surface. Experiments on deliberately injured muscle showed that some of these factors are associated with BL; when muscles were denervated, damaged, and then x-irradiated to prevent muscle regeneration, axons reinnervated original synaptic sites on the surviving BL sheaths (23Sanes J.R. Marshall L.M. McMahan U.J. J. Cell Biol. 1978; 78: 176-198Crossref PubMed Scopus (397) Google Scholar). Based in part on this result, several groups searched for BL components selectively associated with synaptic sites. By now, several have been identified, including site-restricted laminin and collagen IV variants, proteoglycans, and growth factors held in place by proteoglycans (Fig.2). A few components, such as the collagen IV α1 and α2 chains, are excluded from synaptic sites, and a third class, including entactin and perlecan, is present both synaptically and extrasynaptically (4Sanes J.R. Semin. Dev. Biol. 1995; 6: 163-173Crossref Scopus (28) Google Scholar, 30Patton B.L. Microsc. Res. Tech. 2000; 51: 247-261Crossref PubMed Scopus (80) Google Scholar,43Sanes J.R. J. Cell Biol. 1982; 93: 442-451Crossref PubMed Scopus (182) Google Scholar, 44Sanes J.R. Engvall E. Butkowski R. Hunter D.D. J. Cell Biol. 1990; 111: 1685-1699Crossref PubMed Scopus (499) Google Scholar, 45Miner J.H. Sanes J.R. J. Cell Biol. 1994; 127: 879-891Crossref PubMed Scopus (351) Google Scholar, 46Patton B.L. Miner J.H. Chiu A.Y. Sanes J.R. J. Cell Biol. 1997; 139: 1507-1521Crossref PubMed Scopus (366) Google Scholar, 47Martin P.T. Scott L.J. Porter B.E. Sanes J.R. Mol. Cell. Neurosci. 1999; 13: 105-118Crossref PubMed Scopus (54) Google Scholar, 48Reist N.E. Magill C. McMahan U.J. J. Cell Biol. 1987; 105: 2457-2469Crossref PubMed Scopus (141) Google Scholar, 49Goodearl A.D. Yee A.G. Sandrock Jr., A.W. Corfas G. Fischbach G.D. J. Cell Biol. 1995; 130: 1423-1434Crossref PubMed Scopus (79) Google Scholar). It is still not clear which if any of these components are responsible for selective reinnervation of synaptic sites, but several have now been shown to influence pre- and postsynaptic differentiation. When axons innervate myotubes, they form nerve terminals that contain clusters of neurotransmitter-filled synaptic vesicles and membrane-associated release sites called active zones (5Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1209) Google Scholar). Importantly, these presynaptic specializations occur only in the tiny fraction of the axon that directly contacts the postsynaptic cell, indicating that myotube-derived factors organize presynaptic differentiation. Portions of axons contacting BL sheaths from which muscle fibers had been removed (see above) also acquired active zones and synaptic vesicles as well as the ability to recycle vesicles when electrically stimulated. Moreover, new active zones formed in these terminals precisely in register with struts of BL that marked sites where junctional folds (and active zones) had once been (23Sanes J.R. Marshall L.M. McMahan U.J. J. Cell Biol. 1978; 78: 176-198Crossref PubMed Scopus (397) Google Scholar). This association (Fig. 2) showed that some organizers of presynaptic differentiation were contained within the BL. Among the muscle-derived organizers of presynaptic differentiation are the synaptic laminins. The laminin β2 chain was initially identified by virtue of its concentration in synaptic BL (50Hunter D.D. Shah V. Merlie J.P. Sanes J.R. Nature. 1989; 338: 229-234Crossref PubMed Scopus (447) Google Scholar). Myotubes are able to target β2 to postsynaptic specializations (51Martin P.T. Ettinger A.M. Sanes J.R. Science. 1995; 269: 413-416Crossref PubMed Scopus (81) Google Scholar), leading to formation of a BL in which synaptic sites bear primarily β2-containing trimers whereas extrasynaptic regions are enriched in β1-containing trimers. Moreover, β2 fragments or β2-containing laminin-11 causes motor axons in vitro to stop growing and to start differentiating into nerve terminals (52Porter B.E. Weis J. Sanes J.R. Neuron. 1995; 14: 549-559Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 53Son Y.J. Patton B.L. Sanes J.R. Eur. J. Neurosci. 1999; 11: 3457-3467Crossref PubMed Scopus (28) Google Scholar). This behavior contrasts with the robust neurite outgrowth that β1-containing trimers promote. Together these results suggested a rationale for the existence of multiple laminins; they generate local functional diversity (here, synaptic versus extrasynaptic) in a common structural framework. In direct support of this model, presynaptic differentiation is aberrant at neuromuscular junctions in β2 "knock-out" mutant mice: few active zones form, transmitter release is decreased, Schwann cell processes invade the synaptic cleft, and animals die of neuromuscular weakness around the time of weaning (Fig.3B) (54Noakes P.G. Gautam M. Mudd J. Sanes J.R. Merlie J.P. Nature. 1995; 374: 258-262Crossref PubMed Scopus (403) Google Scholar, 55Patton B.L. Chiu A.Y. Sanes J.R. Nature. 1998; 393: 698-701Crossref PubMed Scopus (117) Google Scholar). Thus, β2 laminins qualify as muscle-derived organizers of presynaptic differentiation. On the other hand, the fact that presynaptic differentiation proceeds to a considerable extent in the absence of β2 indicates that additional organizers exist. Additional analysis of muscle laminins revealed the presence of three α chains in synaptic BL (laminin α2, α4, and α5) but only one (α2) extrasynaptically (46Patton B.L. Miner J.H. Chiu A.Y. Sanes J.R. J. Cell Biol. 1997; 139: 1507-1521Crossref PubMed Scopus (366) Google Scholar). Thus, synaptic BL may contain laminins-4, -9, and -11 (α2β2γ1, α4β2γ1, and α5β2γ1), all of which might be involved in presynaptic differentiation. Genetic studies and analyses in vitro suggest distinct roles for each trimer (Fig. 3, B–D). Laminin-11 promotes presynaptic differentiation and repels Schwann cell processes; laminin-9 promotes the precise alignment of pre- and postsynaptic specializations; and laminin-4 may be important for structural integrity, as is α2-containing laminin-2 extrasynaptically (30Patton B.L. Microsc. Res. Tech. 2000; 51: 247-261Crossref PubMed Scopus (80) Google Scholar, 46Patton B.L. Miner J.H. Chiu A.Y. Sanes J.R. J. Cell Biol. 1997; 139: 1507-1521Crossref PubMed Scopus (366) Google Scholar, 56Patton B.L. Cunningham J.M. Thyboll J. Kortesmaa J. Westerblad H. Edstrom L. Tryggvason K. Sanes J.R. Nat. Neurosci. 2001; 4: 597-604Crossref PubMed Scopus (163) Google Scholar, 57Edwards J.P. Hatton P.A. Wareham A.C. Brain Res. 1998; 788: 262-268Crossref PubMed Scopus (12) Google Scholar). Thus, three members of the same gene family collaborate to promote, organize, and maintain presynaptic differentiation. The distinct activities of synaptic laminins suggest that they have multiple receptors on axons and Schwann cells. Receptors presumably include integrins, which bind laminins generally; indeed, integrin α3 is concentrated at active zones (27Cohen M.W. Hoffstrom B.G. DeSimone D.W. J. Neurosci. 2000; 20: 4912-4921Crossref PubMed Google Scholar). In addition, laminins-9 and -11 co-purify with distinct presynaptic membrane components, the calcium channels that trigger transmitter release and the vesicle-associated protein, SV2, respectively (58Son Y.J. Scranton T.W. Sunderland W.J. Baek S.J. Miner J.H. Sanes J.R. Carlson S.S. J. Biol. Chem. 2000; 275: 451-460Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 59Sunderland W.J. Son Y.J. Miner J.H. Sanes J.R. Carlson S.S. J. Neurosci. 2000; 20: 1009-1019Crossref PubMed Google Scholar). These associations raise the possibility that laminins could organize presynaptic differentiation in part by direct interactions with critical components of the release apparatus. Acetylcholine receptors (AChRs) are diffusely distributed in newly formed myotubes but highly concentrated in the postsynaptic membrane of adult muscle (∼10,000/μm2 synapticallyversus <10/μm2 extrasynaptically). Myotubes can cluster diffuse AChR clusters on their own, but classical studies demonstrated a striking ability of ingrowing axons to organize postsynaptic specializations, including AChRs, precisely at sites of nerve-muscle contact (5Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1209) Google Scholar, 60Sanes J.R. Lichtman J.W. Nat. Rev. Neurosci. 2001; 2: 791-805Crossref PubMed Scopus (793) Google Scholar). Once formed, synaptic specializations are stable. Aggregates of AChRs, associated with synaptic cytoskeletal, transmembrane, and BL components, persist at synaptic sites for many weeks following denervation. The stability of BL suggested that it might play a role in maintaining postsynaptic integrity, and experiments on BL sheaths supported this idea. When myotubes regenerated in these sheaths, following damage and denervation (see above), new postsynaptic specializations, including AChRs, formed in apposition to synaptic BL, even though the axon was absent (61Burden S.J. Sargent P.B. McMahan U.J. J. Cell Biol. 1979; 82: 412-425Crossref PubMed Scopus (224) Google Scholar). These results raised the possibility that some of the nerve-derived organizers of postsynaptic differentiation might be stably maintained in or presented by the BL. In fact, of numerous candidate postsynaptic organizers, only one has unequivocally been shown to play a rolein vivo, and this is a nerve-derived synaptic BL component, z-agrin. Agrin was isolated by McMahan and colleagues (62McMahan U.J. Cold Spring Harbor Symp. Quant. Biol. 1990; 4: 407-418Crossref Scopus (566) Google Scholar) in a search for bioactive components of synaptic BL. Agrin is a heparan sulfate proteoglycan with C-terminal domains that interact with the muscle membrane and an N-terminal domain that mediates binding to laminin in the BL (62McMahan U.J. Cold Spring Harbor Symp. Quant. Biol. 1990; 4: 407-418Crossref Scopus (566) Google Scholar, 63Rupp F. Payan D.G. Magill-Solc C. Cowan D.M. Scheller R.H. Neuron. 1991; 6: 811-823Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 64Denzer A.J. Brandenberger R. Gesemann M. Chiquet M. Ruegg M.A. J. Cell Biol. 1997; 137: 671-683Crossref PubMed Scopus (136) Google Scholar). It is synthesized by motoneurons, transported down axons, and released into the synaptic cleft (48Reist N.E. Magill C. McMahan U.J. J. Cell Biol. 1987; 105: 2457-2469Crossref PubMed Scopus (141) Google Scholar). Loss- and gain-of-function studies support the idea that agrin is necessary and sufficient for postsynaptic differentiation. Targeted deletion of the agrin gene in mice leads to devastating defects in neuromuscular synaptogenesis, and local expression of agrin in denervated muscle leads to assembly of a complete postsynaptic apparatus (65Cohen I. Rimer M. Lomo T. McMahan U.J. Mol. Cell. Neurosci. 1997; 9: 237-253Crossref PubMed Scopus (115) Google Scholar, 66Jones G. Meier T. Lichtsteiner M. Witzemann V. Sakmann B. Brenner H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2654-2659Crossref PubMed Scopus (131) Google Scholar, 67Gautam M. Noakes P.G. Moscoso L. Rupp F. Scheller R.H. Merlie J.P. Sanes J.R. Cell. 1996; 85: 525-535Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar). A potential complication is that muscles as well as motoneurons synthesize agrin. However, only the latter express isoforms generated by inclusion of C-terminal exons called "z"; z-containing isoforms are ≥1000-fold more active than z-minus isoforms at clustering AChRsin vitro, and targeted deletion of just the z exons leads to postsynaptic defects as severe as those seen in the absence of all agrin (68Burgess R.W. Nguyen Q.T. Son Y.J. Lichtman J.W. Sanes J.R. Neuron. 1999; 23: 33-44Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). As a large, multidomain protein, it is not unexpected that agrin interacts with many cellular receptors, including the neural cell adhesion molecules, N-CAM, dystroglycan, and integrins (5Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1209) Google Scholar). Genetic analysis has shown, however, that none of these are required for AChR clustering; instead, the critical receptor of agrin, at least for this function, is a receptor tyrosine kinase called MuSK. Activation of MuSK, in turn, leads to association of AChRs with the cytoskeleton via a cytoplasmic protein called rapsyn (60Sanes J.R. Lichtman J.W. Nat. Rev. Neurosci. 2001; 2: 791-805Crossref PubMed Scopus (793) Google Scholar). By binding agrin, the BL both localizes the signal and allows its persistence delivered by the nerve. In addition, dystroglycan and proteins associated with it are involved in the maturation and maintenance of the postsynaptic membrane in adult animals (60Sanes J.R. Lichtman J.W. Nat. Rev. Neurosci. 2001; 2: 791-805Crossref PubMed Scopus (793) Google Scholar, 69Akaaboune M. Grady R.M. Turney S. Sanes J.R. Lichtman J.W. Neuron. 2002; 34: 865-876Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar); it seems likely that the dystroglycan ligands in synaptic BL, agrin and laminin, are involved in regulating the dynamic stability of the synapse. The BL of skeletal muscle plays a remarkable range of roles during development and in adults. None is understood in detail, but all have been documented convincingly, and molecular analysis is now well underway. Muscle emerges, therefore, as one of the tissues in which we are best able to relate the molecular architecture of BL to its function. Some tentative conclusions, which may be applicable to other tissues, are as follows. 1) The original view of the BL as a strictly mechanical support has been augmented (but not replaced) by the realization that it also has organizing and inductive functions mediated by individual components. 2) The major components of BL, laminins and collagens IV, are not only structural proteins, which form networks within BL and links to neighboring structures, but they are also signaling molecules that activate signal-transducing receptors in the membrane. 3) Both laminins and collagens IV are families of molecules that cells can target to particular domains within a single BL. This diversity provides a means for fine localization of signals within a uniform structural framework. 4) Binding sites on the core BL components mediate association of less abundant components such as proteoglycans. The glycosaminoglycan components of the proteoglycans, in turn, bind, concentrate, and present nominally soluble signaling molecules, such as growth factors. To the structural and inductive roles of BL can therefore be added its ability to serve as a "molecular bulletin board" in which adjoining cells can post messages that direct the differentiation and function of the underlying cells.
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