Karen K. McKee

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 α1-, β1-, or γ1-LN domains or with duplicated β1- or α1-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 α-, β-, and γ-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.

Laminin–sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts

Endoneurial laminins (Lms), β1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non–BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

A laminin 511 matrix is regulated by TAZ and functions as the ligand for the α6Bβ1 integrin to sustain breast cancer stem cells

Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 matrix that promotes self-renewal and tumor initiation by engaging the α6Bβ1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the α5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.

Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the α4 and α5 subunits are expressed in α2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (αLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. αLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert α-short arm deletion-mutant laminins LmΔαLN and LmΔαLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmΔLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmΔLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the αLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states.

α6β1 and α7β1 Integrins Are Required in Schwann Cells to Sort Axons

During development, Schwann cells extend lamellipodia-like processes to segregate large- and small-caliber axons during the process of radial sorting. Radial sorting is a prerequisite for myelination and is arrested in human neuropathies because of laminin deficiency. Experiments in mice using targeted mutagenesis have confirmed that laminins 211, 411, and receptors containing the β1 integrin subunit are required for radial sorting; however, which of the 11 α integrins that can pair with β1 forms the functional receptor is unknown. Here we conditionally deleted all the α subunits that form predominant laminin-binding β1 integrins in Schwann cells and show that only α6β1 and α7β1 integrins are required and that α7β1 compensates for the absence of α6β1 during development. The absence of either α7β1 or α6β1 integrin impairs the ability of Schwann cells to spread and to bind laminin 211 or 411, potentially explaining the failure to extend cytoplasmic processes around axons to sort them. However, double α6/α7 integrin mutants show only a subset of the abnormalities found in mutants lacking all β1 integrins, and a milder phenotype. Double-mutant Schwann cells can properly activate all the major signaling pathways associated with radial sorting and show normal Schwann cell proliferation and survival. Thus, α6β1 and α7β1 are the laminin-binding integrins required for axonal sorting, but other Schwann cell β1 integrins, possibly those that do not bind laminins, may also contribute to radial sorting during peripheral nerve development.

Renal collecting system growth and function depend upon embryonic γ1 laminin expression

In order to understand the functions of laminins in the renal collecting system, the Lamc1 gene was inactivated in the developing mouse ureteric bud (UB). Embryos bearing null alleles exhibited laminin deficiency prior to mesenchymal tubular induction and either failed to develop a UB with involution of the mesenchyme, or developed small kidneys with decreased proliferation and branching, delayed renal vesicle formation and postnatal emergence of a water transport deficit. Embryonic day 12.5 kidneys revealed an almost complete absence of basement membrane proteins and reduced levels of α6 integrin and FGF2. mRNA levels for fibroblast growth factor 2 (FGF2) and mediators of the GDNF/RET and WNT11 signaling pathway were also decreased. Furthermore, collecting duct cells derived from laminin-deficient kidneys and grown in collagen gels were found to proliferate and branch slowly. The laminin-deficient cells exhibited decreased activation of growth factor- and integrin-dependent pathways, whereas heparin lyase-treated and β1 integrin-null cells exhibited more selective decreases. Collectively, these data support a requirement of γ1 laminins for assembly of the collecting duct system basement membrane, in which immobilized ligands act as solid-phase agonists to promote branching morphogenesis, growth and water transport functions.

Perlecan is recruited by dystroglycan to nodes of Ranvier and binds the clustering molecule gliomedin

Dystroglycan promotes nodogenesis in part through recruitment of perlecan to nodes of Ranvier, where it binds to gliomedin and may thereby promote sodium channel clustering.

Conjugation of LG Domains of Agrins and Perlecan to Polymerizing Laminin-2 Promotes Acetylcholine Receptor Clustering

Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and βAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.

Chimeric protein repair of laminin polymerization ameliorates muscular dystrophy phenotype

Mutations in laminin &agr;2-subunit (Lm&agr;2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lm&agr;2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of &agr;LNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of &agr;LNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. &agr;LNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in &agr;LNNd-transgenic dy2J mice. In cultured myotubes, we determined that &agr;LNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lm&agr;2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of &agr;LNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.

Linker proteins restore basement membrane and correct LAMA2-related muscular dystrophy in mice

Transgenic expression of two small linker proteins restores muscle function and prolongs life span in a mouse model of LAMA2-related muscular dystrophy. Building a better basement membrane The most common form of congenital muscular dystrophy is caused by mutations in the gene encoding one chain of laminin-211, a basement membrane component. Deleterious muscle function results from the unstable basement membrane and lack of proper connections to the muscle plasma membrane, leading to muscle degeneration. Using a transgenic mouse model of muscular dystrophy, Reinhard et al. studied whether linker proteins could be used to fortify the basement membrane, using laminin-411 as a scaffold. Transgenic mice expressing two linker proteins—a shorter form of agrin and the fusion protein αLNNd, composed of parts of laminin-α1 and nidogen-1—had stable basement membranes, improved muscle function, and prolonged life spans. These proteins could be a missing link for muscular dystrophy therapy. LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is the most frequent form of early-onset, fatal congenital muscular dystrophies. It is caused by mutations in LAMA2, the gene encoding laminin-α2, the long arm of the heterotrimeric (α2, β1, and γ1) basement membrane protein laminin-211 (Lm-211). We establish that despite compensatory expression of laminin-α4, giving rise to Lm-411 (α4, β1, and γ1), muscle basement membrane is labile in LAMA2 MD biopsies. Consistent with this deficit, recombinant Lm-411 polymerized and bound to cultured myotubes only weakly. Polymerization and cell binding of Lm-411 were enhanced by addition of two specifically designed linker proteins. One, called αLNNd, consists of the N-terminal part of laminin-α1 and the laminin-binding site of nidogen-1. The second, called mini-agrin (mag), contains binding sites for laminins and α-dystroglycan. Transgenic expression of mag and αLNNd in a mouse model for LAMA2 MD fully restored basement membrane stability, recovered muscle force and size, increased overall body weight, and extended life span more than five times to a maximum survival beyond 2 years. These findings provide a mechanistic understanding of LAMA2 MD and establish a strong basis for a potential treatment.