Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.

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. Functionblocking antibodies against both ␣7 and ␤1 integrin subunits were found to abolish VFDNFVLK-mediated process extension from cerebellar granule neurons. VFDNFVLK but not its mutant, VSPNGSLK, induced clustering of neuronal ␤1 integrin immunoreactivity. This strongly implicates FD and FV as important structural elements for receptor activation. Moreover, biochemical experiments revealed an association of the ␣7␤1 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 ␣7␤1 integrin mediates a response to tenascin-C and the first to demonstrate a functional role for the ␣7␤1 integrin receptor in CNS neurons.

Current methods to promote growth of cultured neurons use two-dimensional (2D) glass or polystyrene surfaces coated with a charged molecule (e.g. poly-L-lysine (PLL)) or an isolated extracellular matrix (ECM) protein (e.g. laminin-1). However, these 2D surfaces represent a poor topological approximation of the three-dimensional (3D) architecture of the assembled ECM that regulates neuronal growth in vivo. Here we report on the development of a new 3D synthetic nanofibrillar surface for the culture of neurons. This nanofibrillar surface is composed of polyamide nanofibers whose organization mimics the porosity and geometry of the ECM. Neuronal adhesion and neurite outgrowth from cerebellar granule, cerebral cortical, hippocampal, motor, and dorsal root ganglion neurons were similar on nanofibers and PLL-coated glass coverslips; however, neurite generation was increased. Moreover, covalent modification of the nanofibers with neuroactive peptides derived from human tenascin-C significantly enhanced the ability of the nanofibers to facilitate neuronal attachment, neurite generation, and neurite extension in vitro. Hence the 3D nanofibrillar surface provides a physically and chemically stabile cell culture surface for neurons and, potentially, an exciting new opportunity for the development of peptide-modified matrices for use in strategies designed to encourage axonal regrowth following central nervous system injury.

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The protein kinase Chk1 is required for proper arrest of the cell cycle in response to DNA damage. We have previously shown in Schizosaccharomyces pombe, that upon DNA damage, phosphorylation of Chk1 correlates with checkpoint activation and that phosphorylated Chk1 is capable of interacting with the 14-3-3 proteins, Rad24 and Rad25. The interaction between Rad24 and Chk1 is stimulated tenfold after exposure to DNA damaging agents and we postulate that it is an important event in the DNA damage checkpoint response pathway in fission yeast. We identified a stretch of leucine residues as the domain in Chk1 that mediates the interaction with 14-3-3 proteins. Substitution of leucine residues with alanine disrupts the interaction with Rad24 and also prevents Chk1 from becoming phosphorylated in response to DNA damaging agents. Cells expressing the mutants are sensitive to UV radiation. In this study, we also show that Chk1 accumulates in the nucleus in response to DNA damage and this behavior is dependent on Rad24. Interestingly, the 14-3-3 binding domain mutants also fail to localize to the nucleus prompting a search for localization sequences within Chk1. Our investigations have identified the presence of both functional nuclear import and nuclear export sequences encoded in S. pombe Chk1 that, in conjunction with 14-3-3 proteins, may play a prominent role in regulating Chk1 localization and function.