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First published online 29 November 2005
doi: 10.1242/jcs.02685

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Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells

¹ Division of Diabetes, Metabolism and Endocrinology, Lund University, 221 84 Lund, Sweden
² Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Headington, Oxford, OX3 7LJ, UK

View larger version (9K): [in a new window]   Fig. 1. Endocytosis of single vesicles in INS-1 ß-cells. (A) A cell-attached recording where step-wise changes in capacitance (Im/2f) represent exocytosis (upward) and endocytosis (downward) of single vesicles. Transient changes in conductance (Re) during a capacitance step (arrows) result from the formation of fission pores. Step-wise changes in Re upon a capacitance step result from small phase offsets, which were corrected off-line prior to further analysis. (B) The size distribution, shown both as the square root of capacitance (fF1/2) and vesicle diameter, of endocytotic events observed in cell-attached recordings with 2.6 mM Ca2+ in the patch-pipette and 0 mM glucose/0 mM Ca2+ in the bath.

View larger version (15K): [in a new window]   Fig. 2. Glucose stimulation enhances membrane internalisation and increases endocytotic vesicle size in INS-1 cells. Experiments were performed with 2.6 mM Ca2+ in the patch-pipette and 0 mM Ca2+ in the bath. (A) The rate of membrane internalisation is increased when 10 mM glucose is present in the bath. (B) Glucose did not increase the frequency at which endocytotic events occurred. (C) Glucose increased the unitary capacitance of endocytotic steps, reflecting a significant increase in endocytotic vesicle size. *, P<0.05; ***, P<0.001.

View larger version (15K): [in a new window]   Fig. 3. Endocytotic vesicle size is regulated in a bimodal manner by Ca2+ entry through voltage-dependent channels in INS-1 cells. Experiments were performed with 10 mM glucose and 0 mM Ca2+ in the bath, and pipette-Ca2+ was varied. (A) The frequency of endocytotic events was unchanged at different pipette-Ca2+ concentrations. (B) Inclusion of 2.6 mM Ca2+ in the patch-pipette significantly increased the unitary capacitance of endocytotic steps, indicating an increase in vesicle size. This effect was ameliorated by further increasing pipette-Ca2+ to 10 mM. (C) With 2.6 mM Ca2+ in the patch-pipette, inclusion of the L-type Ca2+ channel inhibitor isradipine (israd., 2 µM) or the R-type Ca2+ channel inhibitor SNX-482 (100 nM) prevented the Ca2+-dependent increase in endocytotic vesicle capacitance. ***, P<0.001.

View larger version (16K): [in a new window]   Fig. 4. Endocytotic vesicle capacitance is regulated by direct application of Ca2+ to inside-out excised membrane patches from INS-1 cells. (A) The frequency of endocytotic events in the excised patch was not changed significantly by bath application of Ca2+ (100 nM free, or 0.5 mM). (B) The size distribution, both as the square root of capacitance (fF1/2) and vesicle diameter, of endocytotic events observed in excised-patch recordings in the absence of Ca2+. (C) Bath application of a buffered 100 nM free-Ca2+ solution increased vesicle capacitance. Similar to the cell-attached experiments, this effect was bimodal, and did not occur upon application of 0.5 mM Ca2+. *, P<0.05; ***, P<0.001.

View larger version (18K): [in a new window]   Fig. 5. Analysis of endocytotic fission pore kinetics. In the left panels, the imaginary (Im) conductance component reflects membrane patch capacitance, whereas the real (Re) component reflects the patch conductance. A transient increase in Re immediately prior to endocytosis reflects the endocytotic fission pore. In the right panels, the endocytotic vesicle capacitance (Cv) and fission pore conductance (Gp) were calculated from Im and Re (see Materials and Methods). After formation, the reduction of fission pore conductance reflects either the constriction or lengthening of the pore. The kinetics of this was examined by fitting Gp with a single exponential decay function (solid line). As observed at the arrow marked (i), the fission pore is first detected as Gp decreases to a measurable level. From here, pore conductance decays (marked ii) and finally decreases abruptly to zero (at the arrow marked iii), representing closure of the fission pore and removal of the endocytotic vesicle from the patch membrane as indicated by the simultaneous decrease in Cv.

View larger version (36K): [in a new window]   Fig. 6. Acceleration of membrane fission kinetics by glucose-dependent Ca2+ entry in INS-1 cells. Experiments were performed in the cell-attached configuration. In the bath, Ca2+ was absent and glucose was varied between 0 and 10 mM. In the patch-pipette, Ca2+ was varied and channel inhibitors were included as indicated. Changes in fission pore conductance (Gp) were fit with single exponential decay functions. (A,B) With 2.6 mM Ca2+ in the patch-pipette, glucose stimulation accelerated the kinetics of membrane fission, indicated by a more rapid decrease in fission pore conductance and a faster fission pore time constant (). This glucose-dependent acceleration was prevented by removal of Ca2+ from the patch-pipette, but was not further increased when 10 mM Ca2+ was present. (C,D) In the presence of 10 mM glucose, the acceleration of membrane fission observed with 2.6 mM Ca2+ present in the patch-pipette was prevented by inclusion of the L-type Ca2+ channel inhibitor isradipine (2 µM) or the R-type Ca2+ channel inhibitor SNX-482 (100 nM). *, P<0.05.

View larger version (19K): [in a new window]   Fig. 7. Acceleration of membrane fission by Ca2+ in inside-out excised membrane patches from INS-1 cells. Changes in fission pore conductance (Gp) were fit with single exponential decay functions. Bath application of a 100 nM free-Ca2+ buffer or 0.5 mM Ca2+ accelerated the kinetics of membrane fission. (A) Representative Gp traces with 0 mM Ca2+, 100 nM free-Ca2+ or 0.5 mM Ca2+ in the bath. (B) Fission pore time constants () were calculated by fitting the Gp traces with single exponential decay functions. Unlike what was seen with endocytotic vesicle size, the effect of Ca2+ on fission kinetics was not bimodal. *, P<0.05; **, P<0.01.

View larger version (13K): [in a new window]   Fig. 8. The Ca2+-dependent increase in endocytotic vesicle size, but not the Ca2+-dependent acceleration of fission kinetics, is prevented by inhibition of calcineurin. Experiments were performed on inside-out excised membrane patches from INS-1 cells in the presence of a 100 nM free-Ca2+ buffer. (A) The frequency at which endocytotic events occurred was not changed by the calcineurin inhibitor deltamethrin (1 µM), but was increased in the presence of exogenous purified calcineurin (0.4 U/ml) and calmodulin (100 U/ml). (B) Antagonism of calcineurin with deltamethrin prevented the Ca2+-induced increase in endocytotic vesicle capacitance, whereas exogenous calcineurin/calmodulin had no effect. The control condition in this case represents vesicle capacitance during 100 nM free-Ca2+ stimulation. (C) Neither antagonism of calcineurin with deltamethrin nor exogenous calcineurin/calmodulin significantly affected the rate of membrane fission during 100 nM free-Ca2+ stimulation. *, P<0.05; ***, P<0.001.

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