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 Ca-i(2+) and H-i(+). Fura-2 accumulated in the cytoplasm to a concentration of 50 mu M, whereas BCECF reached a concentration of 200 mu M, The average Ca-i(2+) was estimated to be 180+/-40nM and the average pH(i) 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+ or 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 pH(i) 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.