Direct measurement of cytosolic calcium and pH in living Chlamydomonas reinhardtii cells.
Intracellular free Ca2+ and H+ were quantified in Chlamydomonas reinhardtii, using the fluorescent ion indicators Fura-2 and BCECF. We demonstrate that both indicators can be loaded into living cells as acetoxymethylesters. The esters were hydrolyzed intracellularly to genuine Fura-2 and BCECF capable of indicating changes in Ca2+i and H+i. Fura-2 accumulated in the cytoplasm to a concentration of 50 microM, whereas BCECF reached a concentration of 200 microM. The average Ca2+i was estimated to be 180 +/- 40 nM and the average pHi was 7.4 +/- 0.1. To document the applicability of the ion indicators in Chlamydomonas, we tested their responses to several stimuli. We observed increases in cytoplasmic Ca2+ in response to elevated external Ca2+ on membrane-permeable acids, which are known to induce flagellar excision in Chlamydomonas. The membrane-permeable acids caused a decrease in cytoplasmic pH. Pulses of photosynthetically active light lead to transient pHi changes. Finally, concomitant measurements of rhodopsin-triggered and voltage-sensitive photocurrents indicated that Ca2+ influx is accompanied by a transient depolarisation of the plasmalemma. These experiments document that Fura-2 and BCECF are versatile dyes for studying various ionic processes in Chlamydomonas.
Braun
F J
FJ
Institut für Biochemie I, Universität Regensburg, Germany. Jo.Braun@vkl.uni-regensburg.de
Hegemann
P
P
D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D002118:Calcium / Q000032:analysis*; D002463:Cell Membrane Permeability; D016825:Chlamydomonas reinhardtii / Q000737:chemistry* / Q000502:physiology; D003593:Cytoplasm; D003600:Cytosol / Q000737:chemistry* / Q000502:physiology; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008027:Light; D010788:Photosynthesis
D001565:Benzoates; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007476:Ionophores; D015759:Ionomycin; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D002118:Calcium; D016257:Fura-2