While the interactions of cells with polymeric substrata are widely studied, the influence of cell-cell cohesivity on tissue spreading has not been rigorously investigated. Here we demonstrate that the rate of tissue spreading over a two-dimensional substratum reflects a competition or ''tug-of-war'' between cell-cell and cellsubstratum adhesions. We have generated both a ''library'' of structurally related copolymeric substrata varying in their adhesivity to cells and a library of genetically engineered cell populations varying only in cohesivity. Cell-substratum adhesivity was varied through the poly(ethylene glycol) content of a series of copolymeric substrata, whereas cell-cell cohesivity was varied through the expression of the homophilic cohesion molecules Nand R-cadherin by otherwise noncohesive L929 cells. In the key experiment, multicellular aggregates containing about 600 cells were allowed to spread onto copolymeric surfaces. We compared the spreading behavior of aggregates having different levels of cell-cell cohesivity on a series of copolymeric substrata having different levels of cell-substratum adhesivity. In these experiments, cell-cell cohesivity was measured by tissue surface tensiometry, and cell-substratum adhesivity was assessed by a distractive method. Tissue spreading was assayed by confocal microscopy as the rate of cell emigration from similar-sized, fluorescencelabeled, multicellular aggregates deposited on each of the substrata. We demonstrate that either decreasing substratum adhesivity or increasing cell-cell cohesivity dramatically slowed the spreading rate of cell aggregates.T issue spreading over a substratum is a fundamental process in animal development, wound healing, and malignancy. Understanding the process of tissue spreading is critically important for the emerging field of tissue engineering and for the future use of biomaterials scaffolds for tissue or organ regeneration. Because spreading of a three-dimensional tissue over a surface must come at the expense of associations between cells, greater cell-cell cohesivity should restrain cell emigration (1). Evaluating the expectation that tissue spreading involves a competition between cell-cell and cell-substratum attachments requires the isolation of these two variables from all others. Toward that end, a ''library'' of four cell lines identical except in their cadherin expression and consequently in their cohesivity has been generated. To vary the cell-substratum adhesivity, a library of six structurally related copolymers differing in poly-(ethylene glycol) (PEG) content has been prepared. We then compared the adhesion-related behavior of each of these cell populations on each of these substrata. Materials and MethodsReagents. DMEM, antibiotics (penicillin-streptomycin, gentamycin, G418) and culture supplements (sodium pyruvate; nonessential amino acids, L-glutamine) were purchased from BRL Products (Life Technologies, Gaithersburg, MD). FBS was obtained from HyClone. Fibronectin, laminin, trypsin-EDTA, and PKH2 green fl...

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LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.

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