Artificial laminin polymers assembled in acidic pH mimic basement membrane organization

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 +/- 5 and 86 +/- 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin alpha1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.     

Colocalization of multiple laminin isoforms predominantly beneath hemidesmosomes in the upper lamina densa of the epidermal basement membrane.

Multiple laminin isoforms including laminins 5 (alpha3 beta3 gamma2), 6 (alpha3 beta1 gamma1), 10 (alpha5 beta1 gamma1), and possibly laminins 7 (alpha3 beta2 gamma1) and 11 (alpha5 beta2 gamma1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 (alpha3 beta3 gamma2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the alpha5, beta1, beta2, and gamma1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n=15) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the alpha5, beta1, and gamma1 chains (present in laminins 10/11) and beta2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n=6). In the basement membrane of human epidermis, the alpha5, beta1, beta2, and gamma1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the alpha5, beta1, beta2, and gamma1 chains to the upper lamina densa, with between 84% and 92% of labeling restricted to beneath the HDs, similar to laminin 5 (n> or =200 gold particles per sample, sample number n=4) but distinct from collagen IV labeling (with only 63% labeling beneath HDs, p<0.001). Taken together, the majority of the alpha5beta1/beta2gamma1 laminin chains are located beneath HDs. This suggests that laminin-10-associated chains have specific functions or molecular interactions beneath HDs in the epidermal basement membrane.     

Ultrastructural organization of the glomerular basement membrane as revealed by a deep-etch replica method

Summary The fine structure of the glomerular basement membrane was re-evaluated by using a deep-etch replica method.The structure of the laminae rarae interna and externa of the rat glomerular basement membrane was basically identical in that 6 to 8 nm fibrils were interconnected to form a three-dimensional, polygonal network. The size of the mesh was quite variable but most often ranged from 20 to 25 nm in width. In addition, a zipper-like substructure of the epithelial slit diaphragm was observed. By contrast, the lamina densa was composed of closely packed particles.After exposure of the bovine glomerular basement membrane to ultrasonic waves or trypsin, the particles of the lamina densa were effectively removed. The underlying structure showed the fibrillar network closely resembled that seen in the laminae rarae of the rat glomerular basement membrane.The glomerular basement membrane thus revealed was as principally composed of a fibrillar network, which might be regularly arranged units of type-IV collagen. Numerous fine particles, most likely proper components of the glomerular basement membrane, were attached onto this basic fibrillar structure, giving rise to a morphologic appearance different from that of the laminae rarae.     

Defective glomerulogenesis in the absence of laminin alpha5 demonstrates a developmental role for the kidney glomerular basement membrane

Laminins are major components of all basement membranes. They are a diverse group of alpha/beta/gamma heterotrimers formed from five alpha, three beta, and three gamma chains. Laminin alpha5 is a widely expressed chain found in many embryonic and adult basement membranes. During embryogenesis, alpha5 has a role in disparate developmental processes, including neural tube closure, digit septation, and placentation. Here, we analyzed kidney development in Lama5 mutant embryos and found a striking defect in glomerulogenesis associated with an abnormal glomerular basement membrane (GBM). This correlates with failure of the developmental switch in laminin alpha chain deposition in which alpha5 replaces alpha1 in the GBM at the capillary loop stage of glomerulogenesis. In the absence of a normal GBM, glomerular epithelial cells were in disarray, and endothelial and mesangial cells were extruded from within the constricting glomerulus, leading to a complete absence of vascularized glomeruli. In addition, a minority of Lama5 mutant mice lacked one or both kidneys, indicating that laminin alpha5 is also important in earlier kidney development. Our results demonstrate a dual role for laminin alpha5 in kidney development, illustrate a novel defect in glomerulogenesis, and indicate a heretofore unappreciated developmental role for the GBM in influencing the behavior of epithelial and endothelial cells.     

Ultrastructural localization of fibronectin and laminin in the basement membranes of the murine kidney

Affinity-purified rabbit antibodies specific for two large noncollagenous gycoproteins--laminin and fibronectin--were used to study the distribution of these proteins in normal murine kidneys. Immunofluorescence staining of conventional frozen sections demonstrates fibronectin within mesangial areas of the glomerulus. Laminin is also found in mesangial areas. However, it also appears to be distributed in typical basement membranelike patterns on glomerular and tubular basement membranes and Bowman's capsule. At the ultrastructural level, by labeling 600-800-A thick frozen sections with a three-stage procedure consisting of specific antibodies, biotinyl sheep anti-rabbit IgG, and avidin-ferritin conjugates, fibronectin is present ony in the mesangial matrix and is specifically localized to areas immediately surrounding mesangial cell processes. Laminin, on the other hand, is found uniformly distributed throughout tubular basement membranes, the mesangial matrix, and Bowman's capsule. In glomerular basement membranes, laminin labeling is restricted to the lamina rara interna and adjacent regions of the lamina densa.     

Role of laminin terminal globular domains in basement membrane assembly

Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of alpha-, beta-, and gamma-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin.     

Ultrastructural architecture of proteoglycans in the glomerular basement membrane. A cytochemical approach

Rat kidneys were perfused with fixative solutions containing either a) a polycationic dye (Alcian blue 8 GX, Astra blue 6 GLL, cuprolinic blue, ruthenium red), b) a monocationic dye (safranine 0), or c) Alcian blue in the presence of a 0.3 M MgCl2 concentration. Whereas solutions of a revealed the glomerular basement membrane proteoglycans as particles or threads 60 nm apart and arranged in a reticular pattern, solutions of b and c demonstrated new morphological aspects of these molecules. They appeared as tiny filamentous structures, about 100 to 160 nm long, ordered in a network-like pattern with a mesh of about 60-nm width. The filaments displayed lateral branches about 20 nm apart and about 25 nm long, projecting within the meshes. We suggest that the filamentous structures are the protein core, and the branches are the glycosaminoglycans of proteoglycan molecules. Because of this arrangement the negatively charged sites of the glomerular basement membrane would lie closer to each other than previously assumed.     

Ultrastructural development features of the human glomerular basement membrane

In the human embryo, the basement glomerular membrane appears early, at about ten weeks of age. The study of 36 human embryos aged 8-20 weeks revealed many arguments suggesting the epithelial origin of the basement membrane of the visceral capsula.     

The role of the laminin beta subunit in laminin heterotrimer assembly and basement membrane function and development in C. elegans

Laminins are components of basement membranes that are required for morphogenesis, organizing cell adhesions and cell signaling. Studies have suggested that laminins function as alpha(x) beta(y) gamma(z) heterotrimers in vivo. In C. elegans, there is only one laminin beta gene, suggesting that it is required for all laminin functions. Our analysis is consistent with the role of the laminin beta as a subunit of laminin heterotrimers; the same cells express the laminin alpha, beta, and gamma subunits, the laminin beta subunit localizes to all basement membranes throughout development, and secretion of the beta subunit requires an alpha subunit. RNAi inhibition of the beta subunit gene or of the other subunit genes causes an embryonic lethality phenotype. Furthermore, a distinctive set of phenotypes is caused by both viable laminin alpha and beta partial loss-of-function mutations. These results show developmental roles for the laminin beta subunit, and they provide further genetic evidence for the importance of heterotrimer assembly in vivo.     

Loss of laminin epitopes during glomerular basement membrane assembly in developing mouse kidneys.

Kidney glomerular basement membranes (GMBs) originate in development from fusion of a dual basement membrane between endothelial cells and primitive epithelial podocytes. After fusion, segments of newly synthesized matrix, derived primarily from podocytes, appear as subepithelial outpockets and are spliced into GBMs during glomerular capillary loop expansion. To investigate GBM assembly further, we examined newborn mouse kidneys with monoclonal rat anti-mouse laminin IgGs (MAb) conjugated to horseradish peroxidase (HRP). In adults, these MAb strongly label glomerular mesangial matrices but bind only weakly or not at all to mature GBMs. In contrast, anti-laminin MAb intensely bound newborn mouse GBMs undergoing initial assembly. After intraperitoneal injection of MAb-HRP into neonates, dense binding occurred across both subendothelial and subepithelial pre-fusion GMBs as well as forming mesangial matrices. Considerably less MAb binding was seen, however, in post-fusion GBMs from more mature glomeruli in the same section, although mesangial matrices remained positive. In addition, new subepithelial segments in areas of splicing were negative. These results conflict with those obtained previously with injections of polyclonal anti-laminin IgGs into newborns or adults, which result in complete labeling of all GBMs. Although epitope masking cannot be completely excluded, we believe that decreased MAb binding to developing GBM reflects actual epitope loss. This loss could occur by laminin isoform substitution, conformational change, and/or proteolytic processing during GBM assembly.     

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.     

Fibronectin and laminin in the extracellular matrix and basement membrane of sea urchin embryos

Fibronectin and laminin have been found in the extracellular matrix and in the basement membrane of sea urchin embryos during early development. These glycoproteins are also found on the cell surfaces of the outer epithelial layer and on the secondary mesenchyme cells within the blastocoel. The similarity of functions of the extracellular matrix and basement membrane is discussed, as is the similarity of their molecular components. These observations suggest the possibility that fibronectin and laminin form a continuous matrix surrounding the cells which links the outer ECM (hyaline layer) to the inner ECM (basement membrane). Such a network could coordinate the various activities of the embryo during early morphogenesis.     

Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the glomerular basement membrane

In developing glomeruli, laminin alpha5 replaces laminin alpha1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin alpha5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin alpha5 domains VI through I fused to the human laminin alpha1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 -/- background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 -/- glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin alpha5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the alpha5 G domain essential for mesangial cell adhesion to alpha5LG3-5. Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.     

The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.     

Lipid Rafts & Co.: an integrated model of membrane organization in T cell activation

The model of membrane compartmentalization by self-organizing functional lipid microdomains, named lipid rafts, has been a fruitful concept resulting in great progress in understanding T cell signal transduction. However, due to recent results it has become clear that lipid rafts describe only one out of several membrane organizing principles crucial for T cell activation besides fences and pickets and protein-protein interactions that take part in the formation of the immunological synapse as a highly organized structure at the T cell contact site to the antigen-presenting cell. This review describes the concepts of lipid rafts and other membrane organizing principles to evolve a novel integrated model on the functional role of microdomains in immunological synapse formation and T cell activation. Further research has to elucidate the relative contribution and interrelation of different modes of membrane organization in productive T cell activation.     

Oxytalan fibres in the epidermal basement membrane of Lineus atrocaeruleus (Nemertia)

The basement membrane of the epidermis in Lineus atrocaeruleus (Schmarda 1859) (Nemertia: Heteronemertea) is formed by a tight network of oxytalan fibres which send projections into the epidermis and branch towards the proximal dermis, in close association with the subepidermal musculature. The histophysiology of this relationship is discussed.     

Migratory interaction of amphibian epidermal cells with components of the basement membrane

In adult newts, basal epidermal cells adjacent to a fresh wound move toward the damaged area by migrating over the epidermal basement membrane. In an attempt to determine which basement membrane components mediate this migration, small pieces of glass coated with various natural matrices, purified proteins, or fragments of proteins were implanted into skin wounds such that epidermal cells attempting to form a wound epithelium would encounter the implants. Laminin derived from a cell line (M1536-B3) that produces no type IV collagen was inactive as a migration substrate. Migration on recombinant entactin was somewhat better than on laminin but was still only ～ 14% of that on type I collagen. M15 matrix, a laminin and entactin-containing product of M1536-B3 cells, was no better than entactin alone. Type IV collagen was an excellent substrate, producing slightly more migration than corresponding concentrations of type I collagen at nearly all concentrations tested. Migration on type IV lacking the NC1 domain was at least as good as on intact type IV. All the activity in type IV was present in a 95 kD fragment (al (IV)95) from the carboxy terminal two-thirds of the α1 chain. Approximately 60% of the activity on β1(IV)95 was obtained on implants coated with a 110 amino acid fragment of the α1 chain derived from the carboxy terminal half of α1(IV)95. Adding the synthetic peptide, arg-gly-asp-ser (RGDS) to the medium, biocked migration on fibronectin-coated implants but had no effect on implants coated with type IV, suggesting that migration on type IV involves different cell surface receptors than those mediating migration over fibronectin. Matrigel, a commercial product containing most basement membrane components, was a poor migration substrate. Thus if type IV mediates basal cell migration toward a wound in vivo, there may have to be some alterations in basement membrane structure to allow epidermal receptors to access type IV active site(s). © 1994 Wiley-Liss, Inc.     

Macromolecular organization of collagen fibres in natural and tanned basement membrane

The elastic constants and ultrastructure of natural and tanned basement membrane of the crystalline lens of the adult rat have been investigated. Sonicated and negatively stained specimens of both membranes show parallel filaments that have similar spacing of 3.5(+/- 0.1) nm and a different periodicity. In natural membrane the periodicity is 3.7(+/- 0.13) nm, whilst in tanned basement membrane the periodicity is 3.2(+/- 0.15) nm. The periodicity ratio of tanned membrane to natural membrane was 0.86 +/- 0.04, whilst the elongation ratio of tanned membrane compared with natural membrane was 0.88 +/- 0.05. In contrast to this, the thickness ratio of tanned to natural membrane was 1.098 +/- 0.045. Tanned basement membrane showed a shrinkage of 12% in length but an increase in thickness of about 10%. These data suggest, firstly, that the degree of extension of the superhelices of the filaments follows closely the degree of extension of the intact membrane and, secondly, that the coiled superhelices of tanned membrane have an angle of tilt of about 42 degrees compared with those of natural membrane, where the angle is about 50 degrees. The Young's modulus of elasticity and ultimate stress of tanned basement membrane are, respectively, eight times greater and one-third as great as natural membrane. The entropy change in basement membrane was calculated from the external work necessary to extend the tanned membrane, and was estimated to be -13.5(+/- 2.4) J K-1 mol-1. An estimate of the change in entropy from thermodynamic measurements made on a suspension of collagen tanned with glutaraldehyde was found to be -30.1(+/- 9.5) J K-1 mol-1. The two different estimates of the change in entropy of collagen following tanning suggest that in basement membrane only about 45% of the collagenous protein has an extensile helical structure.     

Intestinal basement membrane of Ascaris suum. Molecular organization and properties of the collagen molecules

The collagenous components of Ascaris suum intestinal basement membrane were isolated by extraction with 0.1 M Tris-HC1, 0.5 M NaC1, 0.5% 2-mercaptoethanol, pH 8.3, and Sephacryl S-300 gel filtration. Rotary-shadowing electron microscopy showed that the collagenous components occur as monomers and dimers with mean contour lengths of 469 +/- 21 and 918 +/- 24 nm, respectively. The molecules each contain a globular domain, with that of the dimer being slightly larger than that of the monomer. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions revealed two polypeptides of Mr = 185,000 and 179,000. A similarity to type IV collagen was indicated by a glycine content of less than 33 mol % and the presence of fucose, mannose, and glucosamine residues. Treatment of the collagen with pepsin resulted in loss of the globular domains but retention of 90% of the length of fibrous collagen segments. Collagenase, however, removed the fibrous regions but left the globular moieties intact. These results extend the previously proposed model (Hung, C.-H., Noelken, M. E., and Hudson, B. G. (1981) J. Biol. Chem. 256, 3822-3826) in which the collagenous domain consists of two monomer-sized triple-helical subunits joined end-to-end by disulfide bonds, with the constituent chains of each subunit being cross-linked by disulfide bonds.