Confocal microscopy analysis of the roles of intracellular pH in the regulation of polarised growth of Dryopteris protonemata

• Main Author: Parton, Richard M.
• Published: University of Edinburgh 1996
• Subjects: 571.2
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.660378

The roles of pH and [Ca<SUP>2+</SUP>] were investigated in relation to the maintenance and regulation of polarity in tip-growing <I>cells of Dryopteris affinis </I>protonemata. Intracellular [ion] was analysed by confocal microscopy of ion-sensitive dyes and ratio imaging. Initially, physiological and cytological aspects of tip growth in apical chlorocytes and rhizoid cells were examined. Findings are discussed in relation to the suitability of protonemata for the study of tip growth and with respect to other commonly studied cell types exhibiting polarised growth. Secondly, the suitability of different methods of introducing ion-sensitive dyes into cells were assessed. Calcium-sensitive dyes failed to load by any method except microinjection, which could not be used routinely because of the poor recovery of cells after injection and rapid vacuolar internalisation of injected dye free-acids. The pH sensitive dyes BCECF and carboxySNARF-1 could both be loaded into cells as their cell permeant AM esters without detrimental effects on cell health. However, sequestration within organelles, particularly the vacuole, proved to be a significant limitation. The problems of dye localisation were compensated for to a certain degree by the use of confocal microscopy and ratiometric analysis. Finally, cytoplasmic pH was examined in growth rhizoids by confocal ratio imaging of AM ester-loaded cSNARF-1. The limits of spatial resolution and precision of pH measurement by this method were estimated at ~ 1μm<SUP>2</SUP> and ~ 0.1 pH unit, respectively (over the pH range 6.9 to 7.3). For rhizoids, average cytoplasmic pH was estimated at 7.1 - 7.3, based on <I>in vitro</I> calibration. No significant cytoplasmic pH gradient (ΔpH of >0.1 unit) was found to be associated with tip growth.