The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Non-canonical Fibroblast Growth Factor Receptor 1 (FGFR1) Interaction Reveals Regulatory and Activating Domains of Neurofascin

Fibroblast growth factor receptors (FGFRs) are important for many different mechanisms, including cell migration, proliferation, differentiation, and survival. Here, we show a new link between FGFR1 and the cell adhesion molecule neurofascin, which is important for neurite outgrowth. After overexpression in HEK293 cells, embryonal neurofascin isoform NF166 was able to associate with FGFR1, whereas the adult isoform NF186, differing from NF166 in additional extracellular sequences, was deficient. Pharmacological inhibitors and overexpression of dominant negative components of the FGFR signaling pathway pointed to the activation of FGFR1 after association with neurofascin in neurite outgrowth assays in chick tectal neurons and rat PC12-E2 cells. Both extra- and intracellular domains of embryonal neurofascin isoform NF166 were able to form complexes with FGFR1 independently. However, the cytosolic domain was both necessary and sufficient for the activation of FGFR1. Cytosolic serine residues 56 and 100 were shown to be essential for the neurite outgrowth-promoting activity of neurofascin, whereas both amino acid residues were dispensable for FGFR1 association. In conclusion, the data suggest a neurofascin intracellular domain, which activates FGFR1 for neurite outgrowth, whereas the extracellular domain functions as an additional, regulatory FGFR1 interaction domain in the course of development.The four known fibroblast growth factor receptors (FGFRs),² which are targeted by a large family of 22 fibroblast growth factor ligands, represent a highly diverse signaling system important for migration, proliferation, differentiation, and survival of many different cell types (1, 2). fibroblast growth factor activation of FGFR leads to the activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and phospholipase Cγ (PLCγ), depending on the cellular system under study. Non-canonical FGFR interactions with NCAM, cadherins, and syndecan via extracellular domains were also described (1). However, the contribution of intracellular interactions of FGFR1 with further membrane co-receptors is poorly understood. Only cytosolic interaction between FGFRs and EphA4 have been described that are involved in mutual transphosphorylation (3).The cell adhesion molecule neurofascin is important for cell-cell communication in the nervous system (4, 5). Neurofascin regulates many different functions in the brain, suggesting that it functions as a key regulator for both developing and differentiated neural cells. Different alternatively spliced neurofascin isoforms are expressed in different cells and at different times of development (6). Embryonal neurofascin NF166 is important for neurite outgrowth and guidance (7, 8). Recently, a role for neurofascin NF166 for early processes of inhibitory synaptogenesis at the axon hillock and for the positioning of inhibitory synapses at the axon initial segment has been proven (9, 10).In the more developed nervous system, NF166 is replaced by NF186, which is inhibitory for neurite outgrowth (11). NF186 is linked to the cortical actin cytoskeleton via ankyrin_G (12). Clustering of voltage-gated sodium channels both at axon initial segments and at the nodes of Ranvier is conferred by neurofascin NF186 (13, 14). A further cytosolic interaction partner is the PDZ molecule syntenin-1 (15).Despite the well known functional importance of neurofascin in the nervous system, corresponding signaling pathways have not been investigated. In contrast, signaling by the related molecules NCAM and L1 have been studied with regard to the induction of neurite outgrowth in greater detail (for a review, see Refs. 16–18). Both NCAM and L1 induce neurite outgrowth through activation of FGFR1 (19–23). NCAM may further undergo lateral interactions with PrP (prion precursor protein) or GFRα, which is part of the glia-derived neurotrophic factor receptor (24, 25). In addition to FGFR1 interaction, both L1 and NCAM are connected to non-receptor tyrosine kinases. However, whereas NCAM employs the non-receptor kinase c-Fyn as an upstream component, L1 is linked to c-Src (26, 27). L1 converges with NCAM signaling upstream of the MAPK pathway at the level of Raf (18, 21, 28, 29). NCAM may induce alternative signaling pathways, including protein kinase A-dependent signaling or G-proteins (18, 30). NCAM signaling to the nucleus may include activation of CREB and c-Fos or NF-κB (29, 31, 32).Here, we elucidate the molecular mechanisms of neurofascin-FGFR1 interaction for neurite outgrowth. We show that both cytosolic and the extracellular domains are important for the association of FGFR1 with neurofascin. Although the cytosolic domain represents a critical determinant for FGFR1 activation, the extracellular sequences of neurofascin act as a regulator for FGFR1-dependent signal transduction in the course of development.