Expression and localization of a fluorescently tagged t-snare syntaxin 4 in immune cells

Abstract

Mast cells are fascinating, multifunctional, bone marrow derived, tissue-dwelling leukocytes that play a central role in immediate allergic reactions and also can be important as initiators and effectors of innate immunity and adaptive immunity. The trigger for the degranulations of different cytokines and chemokines have specific set of SNARE proteins that have not been fully studied. This study focuses on expression and localization of fluorescently tagged STX4 in mast cells and cloning of SNAP25 in pEGFP. Expression and localization of previously cloned t–SNARE STX4 in pEGFP and pDsRed was studied by using flow cytometry and immunoflourescence microscopy technique and cloning and expression of t-SNARE SNAP25 was done in pEGFP. Expression of STX4 in mast cell line model was observed by western blotting. Confirmation of previously cloned STX4 in pEGFP and pDsRed was done by restriction digestion. Both transfected plasmids into RBL cells showed transfection in flow cytometry. Immunoflourescence microscopy showed pEGFP STX4 localized on plasma membrane but STX4 in pDsREd could not get expressed due to frame shift while cloning. The localization of pEGFP STX4 on plasma membrane was observed clearly at 24 hours. Cloned pEGFP SNAP25 plasmid sequence showed one mutation in 451 position from A to G transition from CTA to CTG and overexpression of transfected RBL cells with pEGFP. SNAP25 localized on plasma membrane. However, the exact role of all these SNARE proteins involved in release of various mast cell mediators is still completely unknown. This study was designed to prepare tools for investigation of roles of t-SNAREs namely STX4 and SNAP25 in mast cell function by microscopic imaging studies in real time. Keywords: Mast cell degranulation, SNAREs, pEGFP, PDsRed, STX4, SNAP25