Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin

The region of tenascin-C containing only alternately spliced fibronectin type-III repeat D (fnD) increases neurite outgrowth by itself and also as part of tenascin-C. We previously localized the active site within fnD to an eight amino acid sequence unique to tenascin-C, VFDNFVLK, and showed that the amino acids FD and FV are required for activity. The purpose of this study was to identify the neuronal receptor that interacts with VFDNFVLK and to investigate the hypothesis that FD and FV are important for receptor binding. Function-blocking antibodies against both alpha7 and beta1 integrin subunits were found to abolish VFDNFVLK-mediated process extension from cerebellar granule neurons. VFDNFVLK but not its mutant, VSPNGSLK, induced clustering of neuronal beta1 integrin immunoreactivity. This strongly implicates FD and FV as important structural elements for receptor activation. Moreover, biochemical experiments revealed an association of the alpha7beta1 integrin with tenascin-C peptides containing the VFDNFVLK sequence but not with peptides with alterations in FD and/or FV. These findings are the first to provide evidence that the alpha7beta1 integrin mediates a response to tenascin-C and the first to demonstrate a functional role for the alpha7beta1 integrin receptor in CNS neurons

Long and short splice variants of human tenascin differentially regulate neurite outgrowth. Mol. Cell Neurosc

Tenascin-C has been implicated in regulation of neurite outgrowth both during development and after injury; however, its role as permissive vs inhibitory remains controversial. We report that different tenascin splice variants may have dramatically different impacts on neuronal growth. In a cell culture model, the largest and smallest splice variants (TN.L and TN.S) of human tenascin both promoted process extension when surface-bound. In contrast, soluble TN.S inhibited outgrowth, whereas soluble TN.L had no inhibitory effect. Perturbation experiments with antibodies, and outgrowth experiments with recombinant tenascin fragments, indicate that the differential properties of these molecules can be attributed to their distinctive array of FN-III repeats. Monoclonal antibodies were used to demonstrate at least two distinct neurite outgrowth promoting domains within the alternatively spliced region. These results suggest that the effect of tenascin on axon growth is a function of splice variants, as well as the form or conformation of those variants

Permeabilization of the plasmalemma and wall of soybean root cells to macromolecules

A technique has been developed that results in the reversible permeabilization of the cell wall and plasmalemma of soybean (Glycine max (L.) Merr.) root cells grown in suspension and callus culture. Cells in culture are treated with saponin (0.1 mg/ml) for 15 min at room temperature. They are then coincubated in separate experiments with fluorescent-derivatized dextrans (20-70 kDa) or fluorescein-conjugated goat anti-rabbit immunoglobulin G to ascertain the exclusion size of macromolecules capable of diffusing across the cell wall and plasmalemma into the cytoplasm. Following an incubation period of 30 min, it was observed by conventional and confocal fluorescence microscopy that all derivatized macromolecules tested (20-140 kDa) could be incorporated into the cytoplasm, but not into the vacuole. This procedure did not appear to affect cell viability adversely. A normal doubling time was observed for these cells following the permeabilization procedure

Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

Cerebral cortical astrocyte responses to polyamide nanofibrillar scaffolds versus poly-L-lysine (PLL)-functionalized planar glass, unfunctionalized planar Aclar coverslips, and PLL-functionalized planar Aclar surfaces were investigated by atomic force microscopy and immunocytochemistry. The physical properties of the cell culture environments were evaluated using contact angle and surface roughness measurements and compared. Astrocyte morphological responses, including filopodia, lamellipodia, and stress fiber formation, and stellation were imaged using atomic force microscopy and phalloidin staining for F-actin. Activation of the corresponding Rho GTPase regulators was investigated using immunolabeling with Cdc42, Rac1, and RhoA. Astrocytes cultured on the nanofibrillar scaffolds showed a unique response that included stellation, cell–cell interactions by stellate processes, and evidence of depression of RhoA. The results support the hypothesis that the extracellular environment can trigger preferential activation of members of the Rho GTPase family, with demonstrable morphological consequences for cerebral cortical astrocytes.National Science Foundation grants PHY-0957776 (VMA and VMT) and ARRA-CBET-0846328 (DIS and IA), the Michigan-Louis Stokes Alliance for Minority Participation Summer Undergraduate Research Academy (AAK), the General Directorate for Higher Education, Ministry of National Education of the Republic of Turkey (VMT), and the New Jersey Commission on Spinal Cord Research Grants 06A-007-SCR1and 06-3058-SCR-E-O (SM)