Screen Strategy
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          To identify new proteins that regulate neurite outgrowth a microscopy-based screening strategy has been developed. We took advantage of human full-length cDNA collection (Wiemann S.B. et al. 2001.) where open reading frames (ORF) were GFP-tagged on their N and C termini. These ORF-GFP fusions have already been characterized according to their subcellular localization in mammalian cells (Simpson J.C. et al. 2000., also check web site: http://gfp-cdna.embl.de/index.html).

          First, changes in subcellular localisation of chosen ORF-GFPs during neurite outgrowth process have been monitored. Proteins that change their localisation or localise to neurite tips are the ones with potential role in the process. Additional functional information has been obtained by overexpressing ORF-GFP and assessing their interference with neurite outgrowth process (see the scheme below).  Measuring how overexpression affects length of the neurites and number of neurites per cell gives first hint of their function. Proteins that affect neurite length are possibly involved in neurite elongation. Proteins that change number of neurites per cell are possibly involved in neurite initiation and polarity establishment by creating a spatial landmark that marks one section of the plasma membrane different from the rest. For those proteins that showed an effect on neurite outgrowth when over-expressed, potential changes in their expression levels in response to NGF stimulation was determined by real-time qRT-PCR (to see the these results follow the link). Protein dynamics during neurite outgrowth was then monitored in living cells by time-lapse microscopy. Finally, the experimental data were integrated into existing knowledge derived from bioinformatics analyses and literature data to propose putative molecular networks involved in neurite outgrowth (to see these results follow the link).

           

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