Dissecting The Functions Of Tetraspanins In Migrasomes Regulation

Migrasome is a new organelle discovered in migrating cells,which may play important roles in inter-cellular signaling.TSPAN4 was identified as a marker protein for migrasome and it is highly enriched on migrasome,but the functions of TSPAN4 during migrasome formation process remains largely unknown.In the present study,we systematically overexpressed all the 33 TSPAN proteins in mammals to observe the changes of migrasomes number in NRK cells.Overexpression of 14 TSPAN proteins can significantly promote migrasome formation,while overexpression of the other 19 TSPAN proteins can not promote migrasome formation.After a screen for cell lines bearing high levels of migrasomes at basal conditions,we chose MGC-803 and L929 cells to observe the effects of TSPAN knockout on migrasome formation.Knockout of TSPAN4 significantly restrains migrasome formation in the MGC-803 cells,but knockout of TSPAN4 or knockdown of CD81 alone did not affect migrasome formation in the L929 cells.Through mutagenesis analysis,we found that deletion of the ABCD domains within the large extracellular loop of TSPAN4,CD82,CD81 and CD151 did not affect their subcellular localization.Overexpression of the ABCD domains deleted TSPAN4 or CD82 can still promote migrasome formation to the levels that is comparable to the wild type proteins,while overexpression of the ABCD domains deleted CD81 or CD151 can strikingly promote more migrasome formation compared to the wild type proteins.A mutation of three conserved polar residues(N,Q and E)in the transmembrane domains of TSPAN4 or CD82 did not affect their subcellular localization,but overexpression of the NQE mutants of TSPAN4 or CD82 promoted much less migrasome formation compared to the wild type proteins.Cholesterol is highly enriched on migrasome,and acute cholesterol depletion by methyl-?-cyclodextrin severely disrupts the migrasomes' and retraction fibers' structure.Migrasome formation was largely reduced when the cells were treated with pravastatin or cultured with lipoprotein-deficient serum medium.In addition,we identified the potential binding proteins of TSPAN4-WT and TSPAN4-?ABCD via immunoprecipitation and mass spectrometry analysis.Preliminary screen results suggest that TMEM106 B and ABHD17 B may also play important roles in the migrasome formation process.Collectively,these results indicate that tetraspanins play important roles in the regulation of migrasome,and shed some light on the molecular mechanism underlying the migrasome formation process.