Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney.

The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.

Nonthermal production of weakly interacting massive particles and the subgalactic structure of the universe.

There is increasing evidence that conventional cold dark matter (CDM) models lead to conflicts between observations and numerical simulations of dark matter halos on subgalactic scales, which rules out the favored candidates for CDM, namely weakly interacting massive particles (WIMPs). We propose a mechanism of nonthermal production of WIMPs and study its implications on the power spectrum. Our results show that, in this context, WIMPs as candidates for dark matter can work well both on large scales and on subgalactic scales.

Agrin binds to the nerve-muscle basal lamina via laminin.

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

Native chick laminin-4 containing the beta 2 chain (s-laminin) promotes motor axon growth.

After denervation of muscle, motor axons reinnervate original synaptic sites. A recombinant fragment of the synapse specific laminin beta 2 chain (s-laminin) was reported to inhibit motor axon growth. Consequently, a specific sequence (leucine-arginine-glutamate, LRE) of the laminin beta 2 chain was proposed to act as a stop signal and to mediate specific reinnervation at the neuromuscular junction (Porter, B.E., J. Weis, and J.R. Sanes. 1995. Neuron. 14:549-559). We demonstrate here that native chick laminin-4, which contains the beta 2 chain and is present in the synaptic basement membrane, does not inhibit but rather promotes motor axon growth. In native heterotrimeric laminin, the LRE sequence of the beta 2 chain is found in a triple coiled-coil region that is formed by all three subunits. We show here that the effect of LRE depends on the structural context. Whereas a recombinant randomly coiled LRE peptide indeed inhibited outgrowth by chick motoneurons, a small recombinant triple coiled-coil protein containing this sequence did not.

Differential neural crest cell attachment and migration on avian laminin isoforms.

A number of laminin isoforms have recently been identified and proposed to exert different functions during embryonic development. In the present study, we describe the purification and partial characterization of several isoforms isolated from chick heart and gizzard, and provide data on the molecular mechanisms underlying the interaction of avian neural crest cells with these molecules in vitro. Laminins extracted from heart and gizzard tissues were separated by gel filtration and purified to homogeneity by sequential lectin and immunoaffinity chromatography by utilizing monoclonal antibodies directed against the avian alpha 2, beta 2 and gamma 1 laminin chains. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern of the polypeptide complexes obtained and immunoblotting with polyclonal antisera allowed the identification of Laminin-2 (alpha 2 beta 1 gamma 1), Laminin-4 (alpha 2 beta 2 gamma 1), and laminins comprising the beta 1, beta 2 and gamma 1 chains associated with a shorter alpha chain which, in SDS-PAGE, co-migrate with the beta/gamma complex in the 200 kDa region. These latter laminins, which are here arbitrarily denoted Laminin-alpha x (heart tissue) and Laminin-G (gizzard tissue), are somewhat distinct in their apparent molecular weight, are differentially associated with nidogen, and appear as "T"-shaped particles similar to Laminin-6 and Laminin-7 when analyzed by transmission electron microscopy following rotary shadowing. In contrast, the avian Laminin-2 and Laminin-4 isoforms exhibit the characteristic cruciform shape described previously for their mammalian counterparts. Isolated neural crest cells differentially attached and migrated on these laminin isoforms, showing a clear preference for Laminin-G. Similarly to the EHS Laminin-1, neural crest cells recognized all avian isoforms through their alpha 1 beta 1 integrin, shown previously to be the primary laminin-binding receptor on these cells. Neural crest cell interaction with the avian laminins was dependent upon maintenance of the secondary and tertiary structure of the molecules, as shown by the marked reduction in cell attachment and migration upon disruption of the alpha-helical coiled-coil structure of their constituent chains. The results demonstrate that different laminin isoforms may be differentially involved in the regulation of neural crest cell migration and suggest that this regulation operates through interaction of the cells with a structurally conserved cell binding site recognized by the alpha 1 beta 1 integrin.

Distinct heparin-binding and neurite-promoting properties of laminin isoforms isolated from chick heart.

Laminin isolated from chick heart is composed of several heterotrimeric variants of 800 and 700 kDa. Here, we used monoclonal antibodies against chick laminin to purify different laminin isoforms from this mixture. Antibody 8D3 specifically removed laminin containing alpha 2 chain from chick heart laminin preparations, leaving behind 700 kDa variants. Using antibody C4 against the laminin beta 2 chain, alpha 2 chain containing variants were further separated into alpha 2 beta 1 gamma 1 and alpha 2 beta 2 gamma 1 laminin, respectively. Laminins containing alpha 2 chain and recognized by antibody 8D3 are cross-shaped molecules. Their expression during embryogenesis is tightly regulated. In 5-day embryos staining with monoclonal antibody 8D3 is restricted to the dermamyotome. Older embryos (8 days) express alpha 2 chain containing variants at myotendinous junction primordia of skeletal muscle, and only late in development these variants are generally expressed in skeletal and heart muscle basement membranes. The 700 kDa laminin variants contain beta 1, beta 2, and gamma 1 subunits affiliated with an immunologically distinct, shorter alpha x chain and appear to be T-shaped in the electron microscope. Whereas laminins with an alpha 2 subunit bind to heparin, variants with the novel alpha x chain do not. Experiments using cultured sympathetic neurons showed that laminins with alpha x chain are less potent than alpha 2 chain containing variants in promoting neurite outgrowth. In contrast, sympathetic neurons cannot discriminate between alpha 2 beta 1 gamma 1 and alpha 2 beta 2 gamma 1 laminin substrates, respectively, and show identical high rates of neurite formation.