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First published online October 7, 2005
Journal of Experimental Biology 208, 3957-3969 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01858

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Electrical properties and fusion dynamics of in vitro membrane vesicles derived from separate parts of the contractile vacuole complex of Paramecium multimicronucleatum

Kazuyuki Sugino^*, Takashi Tominaga, Richard D. Allen and Yutaka Naitoh

Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at Manoa, 2538 The Mall, Honolulu, HI 96822, USA

View larger version (126K): [in a new window]   Fig. 1. Two series of pictures of the contractile vacuole complex-derived membrane vesicles obtained from two different P. multimicronucleatum cells (Ai, Bi). (Aii, Aiii, Bii) The I–V relationships for different membrane vesicles in Ai and Bi. Ai, Bi reveal the portion of the contractile vacuole complex from which each vesicle is derived. n, a microneedle used for incision of the cell or for preventing fusion of the membrane vesicles; p, suction pipette for removing solution around the cell; e, patch-clamp electrode. Scale bars, 50 µm. See the text for details.

View larger version (20K): [in a new window]   Fig. 2. Current responses in a membrane patch of the contractile vacuole complex-derived membrane vesicle obtained from a P. multimicronucleatum cell under on-vesicle patch-clamp mode. The five traces show the membrane currents generated in response to a clamped voltage in the range –80 to +80 mV.

View larger version (64K): [in a new window]   Fig. 3. Changes in the input resistance and the membrane potential of a membrane vesicle derived from the contractile vacuole complex of a P. multimicronucleatum cell accompanied by images of the vesicle's rounding-slackening. (Ai, Bi) Frames of two different vesicles showing rounding and slackening. Rounding of the vesicle is recognizable by the enhanced shadow effect (maximum rounding in Ai is in e and in Bi is in g before bursting in h). (Aii, Bii) Time courses of change in the input resistance, Rm, for the vesicles. (Aiii, Biii) Time courses of change in the membrane potential, Vm, for the vesicles. (a–h) in Ai and Bi correspond to the sequence when each picture was taken, which is also indicated by short vertical bars marked by the letters on the traces for Rm (Aii, Bii) and Vm (Aiii, Biii). e, patch-clamp electrode; n, microneedle; v, membrane vesicle. Scale bars, 50 µm.

View larger version (81K): [in a new window]   Fig. 4. Changes in the volume of membrane vesicles derived from the contractile vacuole complex of P. multimicronucleatum cells produced by a change in the external osmolarity. (Ai) A series of pictures of a vesicle that showed swelling after application of distilled water to the surrounding solution. (Aii) Time course of osmotic swelling of the vesicle shown in Ai. (Bi) A series of pictures of a vesicle that showed shrinkage after application of 3 mol l–1 KCl to the external solution. (Bii) Time course of osmotic shrinkage of the vesicle shown in Bi. Arrows in Aii and Bii show the time when the external osmolarity was changed. Letters a–e on each graph show the times when the pictures in the corresponding frames in Ai and Bi were taken. Vesicle volume is presented as the percentage of that before changing the external osmolarity. Scale bars, 50 µm.

View larger version (91K): [in a new window]   Fig. 5. Swelling of a vesicle derived from the contractile vacuole complex of a P. multimicronucleatum cell that took place after the vesicle had previously decreased to its minimum size in an increased external osmolarity of 1 mol l–1 sorbitol. (A) A series of pictures of the vesicle showing swelling. The white arrow in a points to the shrunken vesicle. (B) Time course of swelling of the vesicle. Vesicle volume is presented as the percentage of its maximum volume in the increased osmolarity. Letters a–j in B show the times when the pictures in the corresponding frames in A were taken. The straight line is the linear approximation of the increase in vesicle volume. Scale bar, 50 µm.

View larger version (197K): [in a new window]   Fig. 6. A series of pictures of four (labelled 1–4 in frame 0) membrane vesicles derived from the contractile vacuole complex of a P. multimicronucleatum cell, showing shrinkage and subsequent fusion when exposed to an increased external osmolarity produced by adding 0.5 mol l–1 sorbitol solution. Numbers on frames correspond to the time (s) when the picture was taken after the start of the increase in external osmolarity. p, a micropipette for application of the sorbitol solution. Scale bar, 10 µm.

View larger version (80K): [in a new window]   Fig. 7. An abrupt slackening in the membrane vesicle (Ai) derived from the contractile vacuole complex and the contractile vacuole (Bi) from P. multimicronucleatum cells. (Aii, Bii) Time course of change in the area of the image of the vesicle and the CV, respectively. Areas are presented as the percentage of the largest area achieved during the maximum degree of slackening prior to CV pore opening during the recording. Insets in Aii and Bii are expanded portions of the time courses presented in red. See the text for the details. Scale bars, 50 µm.