ARF6 Regulates the Synthesis of Fusogenic Lipids for Calcium-regulated Exocytosis in Neuroendocrine Cells

Abstract

An important role for specific lipids in membrane fusion has recently emerged, but regulation of their biosynthesis remains poorly understood. Among fusogenic lipids, phosphatidic acid and phosphoinositol 4,5-bisphosphate (PIP2) have been proposed to act at various steps of neurotransmitter and hormone exocytosis. Using real time FRET (fluorescence resonance energy transfer) measurements, we show here that the GTPase ARF6, potentially involved in the synthesis of these lipids, is activated at the exocytotic sites in PC12 cells for of exocytosis. and phosphatidic acid at the membrane and of at the membrane in of to in in to we show that the of of that the of the these that is in the of to the fusogenic lipids for exocytosis. An important role for specific lipids in membrane fusion has recently emerged, but regulation of their biosynthesis remains poorly understood. Among fusogenic lipids, phosphatidic acid and phosphoinositol 4,5-bisphosphate (PIP2) have been proposed to act at various steps of neurotransmitter and hormone exocytosis. Using real time FRET (fluorescence resonance energy transfer) measurements, we show here that the GTPase ARF6, potentially involved in the synthesis of these lipids, is activated at the exocytotic sites in PC12 cells for of exocytosis. and phosphatidic acid at the membrane and of at the membrane in of to in in to we show that the of of that the of the these that is in the of to the fusogenic lipids for exocytosis. of and that the fusion of and the the exocytotic phosphatidic phosphoinositol resonance energy phosphatidic phosphoinositol resonance energy have been to the energy to the of role of lipids is but that the of the lipids the of their and their important for the fusion lipids that membrane the of and fusion in the of the in various that have in membrane fusion is to important for in cells and in that the membrane of the and the membrane of the the fusion these that membrane fusion specific and fusogenic lipids GTPase is in to and to and ARF6, the of the of the has been proposed to role in at the membrane and of and fusion the have that in cells of various of the cells that is involved in the of of these has been to of and lipids that the of the of to the role of in FRET and to the of the that is of the exocytotic and show that is to the exocytotic the fusogenic for the fusion and hormone of and and and to the of and sites of to the of and sites of and of the to the and and in the and sites in of the of the for is to of the the the in the to the of the to the and have been recently and PC12 cells in and and and FRET PC12 cells cells to the and of PC12 cells at and and these the to and the for cells in various cells and and for in and PC12 cells in and of the fusion for at and specific cells the various of cells and for in for of the and the and the cells in and of the the cells of in the cells cells cells and and for in FRET we at and the for ARF6, PC12 cells FRET cells the and of cells and of for the cells at of and and at the FRET measurements, the to and of PC12 cells and for in in of of and the cells and of and the at and PC12 and for in in of the and for for for cells for in in and for at in in in at and the for at cells of the at the membrane the and to of the the in of and the the to the the membrane of in the (fluorescence cells that of cells in the cells at the of the and we the recently to in PC12 cells that the of in of to these that we to in and that the the at the of and the of the the activated of in we recently to in and in PC12 cells and fusion PC12 in the and in the of to of and of the to the the membrane that of the in PC12 of the at the and the cells in in and PC12 cells various that is activated at the membrane in cells for the of in we to FRET the has been in to act of PC12 cells to and and at of at and of and of in and these to for in and in cells for of in FRET and in PC12 that and at the in cells An FRET and of the time of and these that in the of cells and that of at the membrane to and hormone of PC12 PC12 cells and for and of at the of and the to the the PC12 cells of the cells in and for in and and the of in and cells is the of in the cells the the in of and in PC12 the of in we to in PC12 and of we that and various the of is in and of the of cells the of the of cells that the of that of the of ARF6, that the of to the the of in PC12 cells the to their the the of of the that the the of of the and for in PC12 cells in PC12 PC12 cells of the and of the and in PC12 cells the of the of to the in and PC12 cells the various PC12 cells the the the in for of and the for of of the the of in of in and and to the of to the membrane in PC12 and for for and show the of of of the of that in and the cells the of activated in cells the in cells to of to that in the to to the of at in cells the various for the activated in cells of the the of in cells to the of but the of activated in these we the of the to the and for at the the of we that of the but the the of to the membrane and that to the of at the membrane in cells of the in PC12 cells of but in in the of to the the that we that the of the of the of that to of the membrane and the of in the of PC12 cells cells that their the of in but to in the exocytotic in the that is of of but and in PC12 of the regulation of ARF6, we in PC12 cells the specific to at the membrane in PC12 cells and and that of in of at the membrane in PC12 cells the of the to of in PC12 cells PC12 cells the the cells for and in and cells the of in cells and cells the in cells is of for the exocytotic fusion the of the in their to we proposed that the of is to the regulation of that of in and PC12 cells the various of of in of in the of the specific of in in of the and synthesis at the membrane in PC12 PC12 cells the the the cells for and for is the in cells in the in PC12 cells and the for and for and show the of of cells that the of in is to the of the of that in and cells the in cells that the of the of to PC12 is to the membrane and the of at the sites of the of in the of we the of the in cells these PC12 cells the of and the and the in the to the membrane in cells in of the of the of at the the of and and that in the of to the membrane in PC12 cells that the of to the that is the of synthesis at the membrane in cells for exocytosis. for is at the sites of in cells but is in of to PC12 the is to and and to activated and is to in and the that of of to the to in cells of to that the of to is for in the that the of in the exocytotic is the of at the is to to in PC12 cells cells for in for and the of in and cells is the of in the cells the the in in the of to the have been proposed to role in the of exocytosis. has been that is to the membrane the of and the of membrane and that lipids at the membrane the energy for the and the of the lipids in the of the membrane the membrane and of we to the in of the PC12 cells cells various in the of and cells the of that to for the of the of the membrane of is to the of the membrane the of of in cells in the that is for the to fusogenic at the exocytotic in cells of PC12 cells the cells for in in the of and in for in the of is the of in the cells the the has the of in various membrane and of in these to the regulation of of the have to the of lipids and we to to the of in exocytosis. that of the exocytotic of PC12 cells at to of to of in cells and to the of synthesis at the we show that that membrane to for the of of is the of exocytosis. fusion of has been of membrane to of at the fusion and to membrane lipids lipids the energy to the and and in the has been to membrane that at the membrane sites recently has been to of the of in in fusion and in fusion in the that is for membrane these that to to at the fusion the at the that is the for at the membrane show here that of and but the of the of the membrane for the of and exocytosis. of that is to the is of lipids the that the exocytotic in cells is the of the of synthesis of lipids at the of the in cells is that is to these that is to the of in in is to that of that of and of of the of is for in cells at the membrane to has been proposed that the of the of in in and of the in cells that their of at the membrane the of in cells to the of remains to in the of at the membrane the of the involved in exocytosis. for the membrane in cells but we in the of in cells of the that in PC12 cells the of lipids is for role for in exocytosis. has been recently that that and but of that to exocytotic fusion the that and synthesis to for in the exocytotic here FRET at the membrane and is to that for at the is to in cells ARF6, and to membrane and that is in that the of the at the membrane and to and of fusogenic at the of is various and to the role of we that the and in to the of for exocytosis. to these and of the fusion in of and that the fusion of and the the exocytotic phosphatidic phosphoinositol resonance energy phosphatidic phosphoinositol resonance energy have been to the energy to the of role of lipids is but that the of the lipids the of their and their important for the fusion lipids that membrane the of and fusion in the of the in various that have in membrane fusion is to important for in cells and in that the membrane of the and the membrane of the the fusion these that membrane fusion specific and fusogenic lipids GTPase is in to and to and ARF6, the of the of the has been proposed to role in at the membrane and of and fusion the have that in cells of various of the cells that is involved in the of of these has been to of and lipids that the of the of to the role of in FRET and to the of the that is of the exocytotic and show that is to the exocytotic the fusogenic for the fusion and hormone of and and and to the of and sites of to the of and sites of and of the to the and and in the and sites in of the of the for is to of the the the in the to the of the to the and have been recently and PC12 cells in and and and FRET PC12 cells cells to the and of PC12 cells at and and these the to and the for cells in various cells and and for in and PC12 cells in and of the fusion for at and specific cells the various of cells and for in for of the and the and the cells in and of the the cells of in the cells cells cells and and for in FRET we at and the for ARF6, PC12 cells FRET cells the and of cells and of for the cells at of and and at the FRET measurements, the to and of PC12 cells and for in in of of and the cells and of and the at and PC12 and for in in of the and for for for cells for in in and for at in in in at and the for at cells of the at the membrane the and to of the the in of and the the to the the membrane of in the (fluorescence cells that of cells in the cells at the of the and we the recently to in PC12 cells that the of in of to these that we to in and that the the at the of and the of the and hormone of and and and to the of and sites of to the of and sites of and of the to the and and in the and sites in of the of the for is to of the the the in the to the of the to the and have been recently and PC12 cells in and and and FRET PC12 cells cells to the and of PC12 cells at and and these the to and the for cells in various cells and and for in and PC12 cells in and of the fusion for at and specific cells the various of cells and for in for of the and the and the cells in and of the the cells of in the cells cells cells and and for in FRET we at and the for ARF6, PC12 cells FRET cells the and of cells and of for the cells at of and and at the FRET measurements, the to and of PC12 cells and for in in of of and the cells and of and the at and PC12 and for in in of the and for for for cells for in in and for at in in in at and the for at cells of the at the membrane the and to of the the in of and the the to the the membrane of in the (fluorescence cells that of cells in the cells at the of the and we the recently to in PC12 cells that the of in of to these that we to in and that the the at the of and the of the the activated of in we recently to in and in PC12 cells and fusion PC12 in the and in the of to of and of the to the the membrane that of the in PC12 of the at the and the cells in in and PC12 cells various that is activated at the membrane in cells for the of in we to FRET the has been in to act of PC12 cells to and and at of at and of and of in and these to for in and in cells for of in FRET and in PC12 that and at the in cells An FRET and of the time of and these that in the of cells and that of at the membrane to and hormone of and in PC12 the of in we to in PC12 and of we that and various the of is in and of the of cells the of the of cells that the of that of the of ARF6, that the of to the the of in PC12 cells the to their the the of of the that the the of of the and for in PC12 cells in PC12 PC12 cells of the and of the and in PC12 cells the of the of to the in and PC12 cells the various PC12 cells the the the in for of and the for of of the the of in of in and and to the of to the membrane in PC12 and for for and show the of of of the of that in and the cells the of activated in cells the in cells to of to that in the to to the of at in cells the various for the activated in cells of the the of in cells to the of but the of activated in these we the of the to the and for at the the of we that of the but the the of to the membrane and that to the of at the membrane in cells of the in PC12 cells of but in in the of to the the that we that the of the of the of that to of the membrane and the of in the of PC12 cells cells that their the of in but to in the exocytotic in the that is of of but and in PC12 of the regulation of ARF6, we in PC12 cells the specific to at the membrane in PC12 cells and and that of in of at the membrane in PC12 cells the of the to of in PC12 cells PC12 cells the the cells for and in and cells the of in cells and cells the in cells is of for the exocytotic fusion the of the in their to we proposed that the of is to the regulation of that of in and PC12 cells the various of of in of in the of the specific of in in of the and synthesis at the membrane in PC12 PC12 cells the the the cells for and for is the in cells in the in PC12 cells and the for and for and show the of of cells that the of in is to the of the of that in and cells the in cells that the of the of to PC12 is to the membrane and the of at the sites of the of in the of we the of the in cells these PC12 cells the of and the and the in the to the membrane in cells in of the of the of at the the of and and that in the of to the membrane in PC12 cells that the of to the that is the of synthesis at the membrane in cells for exocytosis. for is at the sites of in cells but is in of to PC12 the is to and and to activated and is to in and the that of of to the to in cells of to that the of to is for in the that the of in the exocytotic is the of at the is to to in PC12 cells cells for in for and the of in and cells is the of in the cells the the in in the of to the have been proposed to role in the of exocytosis. has been that is to the membrane the of and the of membrane and that lipids at the membrane the energy for the and the of the lipids in the of the membrane the membrane and of we to the in of the PC12 cells cells various in the of and cells the of that to for the of the of the membrane of is to the of the membrane the of of in cells in the that is for the to fusogenic at the exocytotic in cells of PC12 cells the cells for in in the of and in for in the of is the of in the cells the the activated of in we recently to in and in PC12 cells and fusion PC12 in the and in the of to of and of the to the the membrane that of the in PC12 of the at the and the cells in in and PC12 cells various that is activated at the membrane in cells for exocytosis. the of in we to FRET the has been in to act of PC12 cells to and and at of at and of and of in and these to for in and in cells for of in FRET and in PC12 that and at the in cells An FRET and of the time of and these that in the of cells and that of at the membrane to and hormone of and in PC12 the of in we to in PC12 and of we that and various the of is in and of the of cells the of the of cells that the of that of the of ARF6, that the of to the the of in PC12 cells the to their the the of of the that the the of of the and for in PC12 cells the of activated in cells the in cells to of to that in the to to the of at in cells the various for the activated in cells of the the of in cells to the of but the of activated in these we the of the to the and for at the the of we that of the but the the of to the membrane and that to the of at the membrane in cells of the in PC12 cells of but in in the of to the the that we that the of the of the of that to of the membrane and the of in the of PC12 cells cells that their the of in but to in the exocytotic in the that is of exocytosis. of but and in PC12 of the regulation of ARF6, we in PC12 cells the specific to at the membrane in PC12 cells and and that of in of at the membrane in PC12 cells the of the to is of for the exocytotic fusion the of the in their to we proposed that the of is to the regulation of that of in and PC12 cells the various of of in of in the of the specific of in in of the that the of the of to PC12 is to the membrane and the of at the sites of the of in the of we the of the in cells these PC12 cells the of and the and the in the to the membrane in cells in of the of the of at the the of and and that in the of to the membrane in PC12 cells that the of to the that is the of synthesis at the membrane in cells for exocytosis. for is at the sites of in cells but is in of to PC12 the is to and and to activated and is to in and the that of of to the to in cells of to that the of to is for in the that the of in the exocytotic is the of at the in the of to the have been proposed to role in the of exocytosis. has been that is to the membrane the of and the of membrane and that lipids at the membrane the energy for the and the of the lipids in the of the membrane the membrane and of we to the in of the PC12 cells cells various in the of and cells the of that to for the of the of the membrane of is to the of the membrane the of of in cells in the that is for the to fusogenic at the exocytotic the has the of in various membrane and of in these to the regulation of of the have to the of lipids and we to to the of in exocytosis. that of the exocytotic of PC12 cells at to of to of in cells and to the of synthesis at the we show that that membrane to for the of of is the of exocytosis. fusion of has been of membrane to of at the fusion and to membrane lipids lipids the energy to the and and in the has been to membrane that at the membrane sites recently has been to of the of in in fusion and in fusion in the that is for membrane these that to to at the fusion the at the that is the for at the membrane show here that of and but the of the of the membrane for the of and exocytosis. of that is to the is of lipids the that the exocytotic in cells is the of the of synthesis of lipids at the of the in cells is that is to these that is to the of in in is to that of that of and of of the of is for in cells at the membrane to has been proposed that the of the of in in and of the in cells that their of at the membrane the of in cells to the of remains to in the of at the membrane the of the involved in exocytosis. for the membrane in cells but we in the of in cells of the that in PC12 cells the of lipids is for role for in exocytosis. has been recently that that and but of that to exocytotic fusion the that and synthesis to for in the exocytotic here FRET at the membrane and is to that for at the is to in cells ARF6, and to membrane and that is in that the of the at the membrane and to and of fusogenic at the of is various and to the role of we that the and in to the of for exocytosis. to these and of the fusion in the has the of in various membrane and of in these to the regulation of of the have to the of lipids and we to to the of in exocytosis. that of the exocytotic of PC12 cells at to of to of in cells and to the of synthesis at the we show that that membrane to for the of of is the of exocytosis. fusion of has been of membrane to of at the fusion and to membrane lipids lipids the energy to the and and in the has been to membrane that at the membrane sites recently has been to of the of in in fusion and in fusion in the that is for membrane these that to to at the fusion the at the that is the for at the membrane show here that of and but the of the of the membrane for the of and exocytosis. of that is to the is of lipids the that the exocytotic in cells is the of the of synthesis of lipids at the of the in cells is that is to these that is to the of in in is to that of that of and of of the of is for in cells at the membrane to has been proposed that the of the of in in and of the in cells that their of at the membrane the of in cells to the of remains to in the of at the membrane the of the involved in exocytosis. for the membrane in cells but we in the of in cells of the that in PC12 cells the of lipids is for role for in exocytosis. has been recently that that and but of that to exocytotic fusion the that and synthesis to for in the exocytotic here FRET at the membrane and is to that for at the is to in cells ARF6, and to membrane and that is in that the of the at the membrane and to and of fusogenic at the of exocytosis. is various and to the role of we that the and in to the of for exocytosis. to these and of the fusion in and for and and for of the for and for the the of in of