Characterization of Intercellular Transport of Ran-GTPase using Bimolecular Fluorescence Complementation (BiFC) system
Student name: Ms Gaurav Geetanjali
Guide: Dr Pallavi Somvanshi
Year of completion: 2016
Host Organisation: NCCS, Pune
Supervisor (Host Organisation): Dr Jomon Joseph
Abstract: Ran (Ras like protein Abundant in Nucleus) is a small GTP binding protein of Ras superfamily, which regulates many processes in the cell like nuclear-cytoplasmic transport, mitotic spindle assembly and post-mitotic envelope assembly. The most important role that Ran plays is in nuclear-cytoplasmic transport of cargoes. Apart from this well-established function, our lab has previously reported that Ran GTPase possesses an ability to get transferred from cell to cell in a GTP and CRM1 (Chromosome Region Maintenance 1) dependent manner. We wanted to establish an easy and robust assay for quantification of this phenomenon and we have employed BiFC (Bimolecular Fluorescence Complementation) for this purpose. For BiFC, which is mostly used for the validation of protein-protein interaction, we have used a two protein component system of Venus protein to which RanQ69L (Non hydrolysable mutant form of RanGTP) and RanBP1 (Ran Binding Protein) are fused with each of the two components respectively. We could visualize the BiFC with the RanQ69L and Ran BP1 system both in fixed condition and also by live unfixed imaging.
Keywords: Ran, CRM1, BiFC (Bimolecular Fluorescence Complementation), RanQ69L, RanBP1
