Myosin II Contributes to Fusion Pore Expansion during Exocytosis

During exocytosis, the fusion pore expands to allow release of neurotransmitters and hormones to the extracellular space. To understand the process of synaptic transmission, it is of outstanding importance to know the properties of the fusion pore and how these properties affect the release process. Many proteins have been implicated in vesicle fusion; however, there is little evidence for proteins involved in fusion pore expansion. Myosin II has been shown to participate in the transport of vesicles and, surprisingly, in the final phases of exocytosis, affecting the kinetics of catecholamine release in adrenal chromaffin cells as measured by amperometry. Here, we have studied single vesicle exocytosis in chromaffin cells overexpressing an unphosphorylatable form (T18AS19A RLC-GFP) of myosin II that produces an inactive protein by patch amperometry. This method allows direct determination of fusion pore expansion by measuring its conductance, whereas the release of catecholamines is recorded simultaneously by amperometry. Here we demonstrated that the fusion pore is of critical importance to control the release of catecholamines during single vesicle secretion in chromaffin cells. We proved that myosin II acts as a molecular motor on the fusion pore expansion by hindering its dilation when it lacks the phosphorylation sites. During exocytosis, the fusion pore expands to allow release of neurotransmitters and hormones to the extracellular space. To understand the process of synaptic transmission, it is of outstanding importance to know the properties of the fusion pore and how these properties affect the release process. Many proteins have been implicated in vesicle fusion; however, there is little evidence for proteins involved in fusion pore expansion. Myosin II has been shown to participate in the transport of vesicles and, surprisingly, in the final phases of exocytosis, affecting the kinetics of catecholamine release in adrenal chromaffin cells as measured by amperometry. Here, we have studied single vesicle exocytosis in chromaffin cells overexpressing an unphosphorylatable form (T18AS19A RLC-GFP) of myosin II that produces an inactive protein by patch amperometry. This method allows direct determination of fusion pore expansion by measuring its conductance, whereas the release of catecholamines is recorded simultaneously by amperometry. Here we demonstrated that the fusion pore is of critical importance to control the release of catecholamines during single vesicle secretion in chromaffin cells. We proved that myosin II acts as a molecular motor on the fusion pore expansion by hindering its dilation when it lacks the phosphorylation sites. Exocytosis is a fundamental cellular mechanism used by neurons and hormone-secreting cells to interact with each other and to influence their environment through the release of neurotransmitters and hormones. These chemical signals are disposed to the extracellular medium in the form of quanta, as vesicles containing transmitter fuse with the plasma membrane and release their cargo. Release occurs through the exocytotic fusion pore, which is the water channel connecting the vesicle interior to the extracellular space. The dynamics of the fusion pore have been mainly investigated at the level of single cells by two techniques: patch clamp measurements of the electrical capacitance of the cell membrane (1Neher E. Marty A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6712-6716Crossref PubMed Scopus (804) Google Scholar, 2Haller M. Heinemann C. Chow R.H. Heidelberger R. Neher E. Biophys. J. 1998; 74: 2100-2113Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 3Dernick G. Alvarez de Toledo G. Lindau M. Nat. Cell Biol. 2003; 5: 358-362Crossref PubMed Scopus (69) Google Scholar) and the amperometric detection of neurotransmitter with carbon fibers (4Leszczyszyn D.J. Jankowski J.A. Viveros O.H. Diliberto Jr., E.J. Near J.A. Wightman R.M. J. Neurochem. 1991; 56: 1855-1863Crossref PubMed Scopus (100) Google Scholar, 5Chow R.H. von Ruden L. Neher E. Nature. 1992; 356: 60-63Crossref PubMed Scopus (722) Google Scholar). Whereas patch clamp detects changes of cell membrane area and conductance caused by vesicular fusion, the electrochemical method analyzes quantitatively the release of catecholamines from each exocytotic event. The combination of whole cell capacitance measurements and amperometry showed that during fusion pore opening there is a small release of neurotransmitter preceding the amperometric spike that is directly proportional to the pore conductance (6Alvarez de Toledo G. Fernandez-Chacon R. Fernandez J.M. Nature. 1993; 363: 554-558Crossref PubMed Scopus (530) Google Scholar). Because then, this signal, the prespike foot (PSF) 2The abbreviations used are: PSF, prespike foot; CA, catecholamine; CFE, carbon fiber electrode; RLC, regulatory light chain; GFP, green fluorescent protein; WT, wild type; PKC, protein kinase C. has been considered an indicative of the lifetime of the early fusion pore (7Amatore C. Arbault S. Bonifas I. Guille M. Lemaitre F. Verchier Y. Biophys. Chem. 2007; 129: 181-189Crossref PubMed Scopus (42) Google Scholar). Later on, higher resolution experiments in rat mast cells by using the patch amperometry technique demonstrated that the size of the fusion pore does not limit release during the upstroke of the amperometric spike (8Tabares L. Lindau M. Alvarez de Toledo G. Biochem. Soc. Trans. 2003; 31: 837-841Crossref PubMed Google Scholar), and it is accepted that the spike phase corresponds to the fast and massive release of transmitters out of the vesicular matrix after the fusion of cell and vesicular membranes. In neurons and neuroendocrine cells it has not been possible to report the fusion pore dynamics during the bulk release of the amperometric spike. The role of the fusion pore has been inferred from amperometric recordings alone (9Barclay J.W. Aldea M. Craig T.J. Morgan A. Burgoyne R.D. J. Biol. Chem. 2004; 279: 41495-41503Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Burgoyne R.D. Fisher R.J. Graham M.E. Haynes L.P. Morgan A. Biochem. Soc. Trans. 2001; 29: 467-472Crossref PubMed Scopus (36) Google Scholar, 11Xu J. Tse F.W. J. Biol. Chem. 1999; 274: 19095-19102Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), suggesting that many protein candidates control the fusion pore by studying the shape of the PSF or amperometric spike (12An S. Zenisek D. Curr. Opin. Neurobiol. 2004; 14: 522-530Crossref PubMed Scopus (68) Google Scholar, 13Wang C.T. Grishanin R. Earles C.A. Chang P.Y. Martin T.F. Chapman E.R. Jackson M.B. Science. 2001; 294: 1111-1115Crossref PubMed Scopus (250) Google Scholar, 14Bai J. Wang C.T. Richards D.A. Jackson M.B. Chapman E.R. Neuron. 2004; 41: 929-942Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Cytoskeletal proteins play an important role organizing the transport of vesicles to release sites (15Dillon C. Goda Y. Annu. Rev. Neurosci. 2005; 28: 25-55Crossref PubMed Scopus (321) Google Scholar). During stimulation, calcium influx induces a dynamic reorganization of the cortical actin network and facilitates exocytosis (16Trifaro J.M. Vitale M.L. Trends Neurosci. 1993; 16: 466-472Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 17Cheek T.R. Burgoyne R.D. FEBS Lett. 1986; 207: 110-114Crossref PubMed Scopus (133) Google Scholar, 18Vitale M.L. Rodriguez Del Castillo A. Tchakarov L. Trifaro J.M. J. Cell Biol. 1991; 113: 1057-1067Crossref PubMed Scopus (177) Google Scholar, 19Giner D. Neco P. Frances Mdel M. Lopez I. Viniegra S. Gutierrez L.M. J. Cell Sci. 2005; 118: 2871-2880Crossref PubMed Scopus (66) Google Scholar). It has been postulated that such transport is based in molecular motors associated with F-actin trails and conduct vesicles along regions with different F-actin organization. More recently, a new role implicating the regulation of single fusion kinetics has been also suggested (20Neco P. Giner D. Viniegra S. Borges R. Villarroel A. Gutierrez L.M. J. Biol. Chem. 2004; 279: 27450-27457Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In that way, myosin II has been postulated to exert a tensional pressure in the F-actin network that could affect membrane tension, fusion pore expansion, or the final extrusion of vesicular contents. In the present work, by using the technique of patch amperometry, we directly determined in chromaffin cells the effect of a protein on the fusion pore dynamics for the first time. This method allowed us to resolve single fusion events from vesicles smaller than 500 nm in diameter by simultaneous measurement of the patch membrane capacitance and the release of catecholamines by placing a carbon fiber detector inside the patch pipette. Single-vesicle exocytosis has been determined in rat chromaffin cells infected by modified herpes virus (amplicons) containing the myosin II regulatory light chain (RLC) or an unphosphorylatable form that produces inactive protein (T18A/S19A RLC) chimeras with green fluorescent protein (GFP). Therefore, by direct measurement of fusion pore conductance by admittance methods, we proved that myosin II plays an essential role in fusion pore expansion during exocytosis. Chromaffin Cell Preparation and Infection—Rat chromaffin cells were obtained from Sprague-Dawley rats (100-200 g) and cultured as described previously (21Gullo F. Ales E. Rosati M. A. L. A. Lopez E. J. 2003; PubMed Scopus Google Scholar). The cells were in modified medium with and The cells were on with and at in an containing with the were infected with a herpes virus containing wild or of obtained as described previously (20Neco P. Giner D. Viniegra S. Borges R. Villarroel A. Gutierrez L.M. J. Biol. Chem. 2004; 279: 27450-27457Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The for the experiments of of after and at along the two amperometry experiments were out after at and and The to and the The and in membrane capacitance and catecholamine release were recorded simultaneously by patch amperometry E. L. J.M. Lindau M. Alvarez de Toledo G. Nat. Cell Biol. 1999; PubMed Scopus Google Scholar, A. G. Alvarez de Toledo G. Lindau M. Nature. PubMed Scopus Google Scholar). patch clamp with a carbon fiber the patch pipette. with two The from carbon fibers as described previously R.H. J. Neher E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The to whereas the to the of a amperometric a The at were with an at were in with a and with a The were and a in the of to when the carbon fiber the We the direct level in the to the of the amperometric We used a patch clamp for capacitance to the of patch admittance were measured as described previously (1Neher E. Marty A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6712-6716Crossref PubMed Scopus (804) Google Scholar, Lindau M. J. Cell Biol. 129: PubMed Scopus Google Scholar) with a using a of at a of The to a or and were with a with pore were as described previously Lindau M. J. Cell Biol. 129: PubMed Scopus Google Scholar). conductance from the and of the admittance after from each single exocytotic the CA, and the area of the to bulk is the to the and the of the The to the and of amperometric spike at its the of release during the fusion pore expansion. spike and capacitance using for for and The for used to the different were considered different when of the were as the from experiments in a of cells. The experiments with cells from at different of Release amperometry measurements from exocytotic events in the patch of membrane the patch pipette. Because of the in patch amperometry amperometric are We the amperometric from a chromaffin from a measured fusion pore by We that the spike is by different release of from the vesicular of through the exocytotic fusion pore, and of the patch for and carbon fiber The used to on the vesicle and the extracellular of in the vesicle M. Heinemann C. Chow R.H. Heidelberger R. Neher E. Biophys. J. 1998; 74: 2100-2113Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The of the and and measured of the fusion pore The that of catecholamines inside the vesicle and in Chem. 1982; Scopus Google Scholar, C. I. L. Chem. J. 1999; 5: Google Scholar). The as a the patch inside the and the at the of the to to amperometry. In each a of by matrix were to in the to through the fusion pore, and to in the The also were to when the cell the or the of the carbon or to when the of the carbon were to to amperometric Release after in the simultaneously membrane fusion and release of catecholamines in chromaffin we have used patch amperometry A. G. Alvarez de Toledo G. Lindau M. Nature. PubMed Scopus Google Scholar). This method a inside the exocytotic events at the membrane patch and their the pipette. In to the simultaneous measurement of capacitance changes caused by single chromaffin and the associated release of patch amperometry allows for a of the dynamics of the fusion pore by admittance We have recorded green fluorescent to infected cells recorded on cells overexpressing the and the unphosphorylatable are shown in and The in patch membrane capacitance of the fusion of single chromaffin with the plasma The amperometric detection with a inside the patch pipette. in capacitance by an amperometric spike in which release of vesicular through the fusion In extracellular amperometry, the whole cell to a of spike caused by In patch amperometry of the events a kinetics on the fusion pore lifetime matrix capacitance changes to in cell area as chromaffin with the plasma We not in the size in and cells cells and These are not different from the size for cells and to that previously in rat chromaffin cells E. L. J.M. Lindau M. Alvarez de Toledo G. Nat. Cell Biol. 1999; PubMed Scopus Google Scholar), that of the form or form of myosin II does not with the or of chromaffin to exocytosis. The the of the capacitance and the of the amperometric spike the for fusion pore expansion and release of experiments showed a the capacitance and the of the amperometric spike in cells the form of myosin II as with the wild protein This effect is the the of the capacitance and the of the spike the the the in cells in suggesting fusion pore expansion bulk are The is the the PSF in the amperometric we could amperometric of the at which the detector is from the cell in patch amperometry experiments were in the of PSF in and cells To the phase of fusion pore we of the and of the The spike and the at the for the unphosphorylatable were by of and with and for wild there in the catecholamine in cells in and in to in the of the that the of vesicle in cells directly on the kinetics of the exocytotic fusion pore, we the of the fusion pore the electrical of a in which the fusion pore is by a conductance in with the vesicle capacitance by the vesicle through this on fusion pore and expansion. The changes in and by of the and of the admittance Lindau M. J. Cell Biol. 129: PubMed Scopus Google Scholar, Neuron. Full Text PDF PubMed Scopus Google Scholar). in and the fusion pore conductance during two exocytotic by release of in a form in and In the the conductance of the pore a of in In the the conductance at a a of In the fusion pore and by method after the The during fusion pore expansion different in and the We not a amperometric foot during fusion pore expansion. the amperometric spike showed a as with the fusion pore In and the of the amperometric spike when the fusion pore conductance the amperometric spike at different of the fusion pore conductance measured in and The of fusion pore conductance for and vesicles are shown in and cells fusion to cells the showed pore than In the lifetime of the fusion pore in cells the has a of and in cells the of The pore conductance obtained in cells and in cells. The of fusion pore expansion as determined from the of in its in and in of the conductance for in cells that than of the conductance a expansion of the fusion pore to to of cells a of conductance The of pore as measured by the in which the is that of the in cells has in in cells a of the in the of and These that the of to a expansion of the fusion pore with conductance to to release the bulk of the To the role of myosin on fusion pore expansion, we the of changes in We the different phases in expansion of the fusion pore during exocytosis. The conductance of fusion in and are shown in the of the fusion pore dynamics from a cell the We at two expansion there is a conductance This early is to a The to with and the phase in cells the the first phase and the expansion phase to than to the cells phase and phase the other during the expansion of the fusion pore the of to higher in the than in cells the wild the also of the phase of pore expansion, the in wild and cells. the of Release during the could fusion pore conductance with the kinetics of neurotransmitter release from the fusion event. it has been that the fusion pore conductance the of catecholamines during foot signals de Toledo Lindau M. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar). it is a of how the fusion pore release during the upstroke of the amperometric spike. To understand how the properties of the fusion affect the we have the pore conductance and the spike in the exocytotic events from the in cells the wild and the We that with than are associated with when the fusion a conductance than 500 the spike at a We the spike with the lifetime of the fusion we a these two in the wild and in the These that the size of the fusion pore and the to the fusion pore are important of the kinetics of release and influence at the phase of the amperometric spike. Because the of the amperometric spike and the spike in the were than in the wild that myosin II fusion pore the that phosphorylation of the the expansion of the fusion pore and, as neurotransmitter and a Release during the of the it has not been possible to the fusion pore conductance during the phase of the amperometric spike from a To the role of fusion pore and myosin II at this we amperometric by of fusion pore expansion in the out different in the exocytotic release through the exocytotic fusion pore, transmitter release from matrix and of the pipette. and were used to and to for the fusion pore expansion and the amperometric spike of different fusion not during exocytosis, that the properties of are To a of fusion pore were used to amperometric the of the of the amperometric spike of fusion pore conductance measured by admittance the conductance measured the of the opening of the fusion pore a that not with the spike when the conductance by in the the fusion pore size and the with the of amperometric Therefore, the fusion pore for the of the amperometric spike that could not measured by admittance the shape of amperometric obtained from the of myosin II by the pore properties when the fusion pore conductance from a and a fusion were used as to the the amperometric showed kinetics to obtained Because it is that myosin II the properties of catecholamines to the it is to that in spike shape is to changes in fusion pore Here, we a mechanism that to the of neurotransmitter a control of the exocytotic fusion This mechanism of in synaptic is determined by the of release of neurotransmitters opening and expansion of the exocytotic fusion The for release in chromaffin cells also important these cells and two hormones for the or the of changes on this for the fusion pore by myosin II not of for the release of We report release of catecholamines to the extracellular medium that in in control and chromaffin cells with the unphosphorylatable form of myosin we are that myosin II to the expansion of the fusion pore during exocytosis. We that the kinetics of release is directly to the of the fusion mechanism has been postulated to control the of release from synaptic vesicles at C. A. M. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). that directly or affect the fusion pore are of the of We have used patch amperometry to vesicle fusion measuring the cell membrane and release of catecholamines in rat chromaffin cells. This method allows a of fusion pore properties and their role in the dynamics of To the role of proteins in fusion pore we infected chromaffin cells with a modified herpes virus (amplicons) to the myosin chimeras with This allows us to myosin II in exocytosis. This to the effect of a form of the protein in exocytosis when this form is that an form of myosin II the fusion and bulk release of This the by the exocytotic to the fusion pore to a size that allows transmitter to the combination of cell membrane capacitance measurements and release from single chromaffin we two The first that release of occurs as with cells in chromaffin cells overexpressing the form of myosin the proved that the pore size at which this is is smaller as with cells. that fusion pore expansion is in which myosin II is not In these are by the that spike are as it has also been shown previously D. Neco P. Frances Mdel M. Lopez I. Viniegra S. Gutierrez L.M. J. Cell Sci. 2005; 118: 2871-2880Crossref PubMed Scopus (66) Google Scholar). using we that the in spike by a fusion pore expansion in cells the form of myosin The effect of myosin on fusion pore expansion by two is that myosin to fusion pore expansion, myosin phosphorylation plays an role in the fusion myosin acts as a in a process. In such way, the of myosin phosphorylation fusion pore expansion. Exocytosis the fusion of to tensional In fusion, of the membrane of is to the of a vesicle a in during exocytosis the and of vesicles to fusion of the vesicle membrane the plasma Therefore, membrane fusion is an in which the fusion pore on the the plasma membrane and vesicle This fusion of smaller vesicles than fusion of vesicles mast cells or mast cells (6Alvarez de Toledo G. Fernandez-Chacon R. Fernandez J.M. Nature. 1993; 363: 554-558Crossref PubMed Scopus (530) Google Scholar). In experiments fusion in chromaffin and Because we are using cell membrane capacitance measurements in the we of to for chromaffin C. S. Alvarez de Toledo G. Rev. 2005; PubMed Scopus Google Scholar). chromaffin cells their fusion pore in each to This could for a process in fusion pore expansion. The molecular this is have been the the of the exocytotic fusion pore M. Curr. Opin. Cell Biol. PubMed Scopus Google Scholar, E.R. Nat. Rev. Biol. PubMed Scopus Google Scholar). In phosphorylation of myosin II with the form of the phases in fusion pore expansion, suggesting that myosin does not play a critical role in the fusion pore acts as a to the kinetics of pore expansion. These to myosin II as of the of the protein that to membrane fusion during exocytosis Fernandez J.M. Curr. Opin. Cell Biol. PubMed Scopus Google Scholar). also that the fusion pore release of at higher which are the limit of the resolution of measured by admittance from and cells studied by that the fusion pore conductance release during exocytosis. In the that fusion in chromaffin that L. Ales E. Lindau M. Alvarez de Toledo G. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) that the or the matrix does not play a critical role in fusion pore expansion at this of the process. the these that the role of myosin II on the exocytosis the expansion process the fusion pore the vesicle membrane the plasma It has been shown that myosin II is associated with the F-actin network and that the associated with of this that control in the of sites D. Neco P. Frances Mdel M. Lopez I. Viniegra S. Gutierrez L.M. J. Cell Sci. 2005; 118: 2871-2880Crossref PubMed Scopus (66) Google Scholar, D. Lopez I. J. Viniegra S. Gutierrez L.M. 2007; PubMed Scopus Google Scholar). In F-actin with demonstrated a of and cortical F-actin in cells overexpressing wild or the unphosphorylatable the to how myosin II the final phases of membrane fusion is that the II network dynamics the fusion pore expansion. Myosin II phosphorylation by the myosin light chain kinase is to the F-actin dynamics vesicle M.L. Rodriguez Del Castillo A. Tchakarov L. Trifaro J.M. J. Cell Biol. 1991; 113: 1057-1067Crossref PubMed Scopus (177) Google Scholar). The of the myosin II by a form in a F-actin network to vesicle D. Neco P. Frances Mdel M. Lopez I. Viniegra S. Gutierrez L.M. J. Cell Sci. 2005; 118: 2871-2880Crossref PubMed Scopus (66) Google Scholar) and pore expansion. In the of myosin phosphorylation on the fusion pore with the influence of protein phosphorylation or on the exocytotic fusion of expansion of exocytotic fusion have been this effect is when is with the kinase S. Lindau M. 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Therefore, as a it is to that different through with or other proteins of the fusion could the of in the exocytotic fusion The combination of molecular and a technique has allowed us to understand how changes in fusion affect neurotransmitter release and new the role of myosin This the to the of many proteins as in exocytosis.