Development of a fluorescent probe for determination of water transport in subcellular organelles.

摘要

Water transport has been widely studied in a variety of cells and isolated membranes. However, the intrinsic complexity related with the study of intracellular processes has delayed the development of an analytical technique for the study of water transport in subcellular compartments of living cells. This study represents the development and application of a ratiometric fluorescent probe to study water transport in endocytic organelles of intact living cells. The probe is based on the use of a D2O-sensitive fluorophore, Lucifer yellow dextran (LY-dex), together with a D2O-insensitive fluorophore, Alexa fluor 546 dextran (AF546-dex), used as an internal reference. To ensure localization of the dyes into the organelles via pinocytocis and avoid leaking, both dyes are coupled to a high molecular weight (10.000 MW) aminodextran.;In the first part of the thesis, demonstration of the efficacy of the probe to measure water transport in living cells is shown. In the second part, the probe is used for quantification of water exchange in lysosomes. Finally, the probe is employed to follow kinetics of water exchange in macropinosomes and lysosomes.;It was demonstrated that the probe responds linearly to changes in D 2O concentration (% v/v). In in vitro experiments the probe ratios changed about 0.40 units between 0% v/v and 90% v/v D2O, while in ex vivo experiments the probe ratio changes about 0.20 units over the same concentration range. To probe its applicability in water transport measurements in living cells, studies of superfusion were conducted replacing the RB (prepared in H2O) around individual cells by Ringer's Buffer containing D2O. Rapid imaging of the organelles during perfusion experiments allowed the determination of kinetics of water exchange across the membranes of lysosomes in CHO cells.;The probe was also used in the quantification of water exchange in J774.A1 lysosomes. It was observed that J774.A1 lysosomes exchange 66 +/- 1% of their water content under equilibrium but when the organelles were permeabilized, using 20 mug/ml digitonin solutions in 90% D2O to increase exchange of water across the lysosomes. Hence, lysosomes exchange the remaining water content.;Taking advantage of the fact that organelles loaded with the fluorescent probe show significant increases in LY-dex fluorescence intensity as D 2O from the extracellular solution is exchanged across the organellar membrane, the probe was used to follow the kinetics of water exchange across the membranes of lysosomes and macropinosomes in J774.A1 cells.;It was found that lysosomal water permeability is pH dependent; changing from (6.8 +/- 0.2) x 10-3 cm/s (mean +/- s.e.m. n=62 lysosomes) to (9.4 +/- 0.5) x 10-4 (mean +/- s.e.m. n=22 lysosomes) following treatment with NH4Cl and (1.3 +/- 0.4) x 10-4 (mean +/- s.e.m. n=4 lysosomes) after treatment with Bafilomycin A1. It was also observed that macropinosome membrane permeability increases from (1.2 +/- 0.6) x 10-3 cm/s (3 min after formation mean +/- s.e.m. n=15 pinosomes) to (5.2 +/- 0.6) x 10-3 cm/s (after 25 min of formation mean +/- s.e.m. n=15 pinosomes).;Collectively these findings demonstrate the ability of the developed methods for quantification and determination of the kinetics of water exchange in subcellular organelles in living cells. This is the first time that kinetics of water and quantification of water has been measured in organelles under physiological conditions.