Lucifer Yellow as a Live Cell Fluorescent Probe for Imaging Water Transport in Subcellular Organelles

Abstract

While the water permeability of the plasma membranes of mammalian cells has been studied extensively, water transport across membranes of subcellular compartments (e.g., lysosomes, macropinosomes) has been difficult to study. Here we demonstrate a new method for measuring water flux in late endosomes and lysosomes of intact living cells using time-lapse fluorescence microscopy. Cells were loaded by fluid-phase uptake with a mixture of the Lucifer Yellow dextran (LY-dex), a D₂O sensitive dye, and a D₂O insensitive control dye, Alexa fluor 546 dextran (AF546-dex). LY-dex responded linearly to changes in D₂O concentration and the LY-dex D₂O sensitivity was not affected by changes in pH, physiological salt, and protein concentrations. The co-loaded control dye, AF546-dex, showed no signal changes as a function of D₂O concentration. To measure membrane water flux, the LY-dex fluorescence in labeled organelles was recorded during rapid superfusion of cells with isotonic buffers prepared in D₂O. The time constant of water exchange across the lysosomal membrane of intact cells was determined by fitting the data to a single exponential function. From these data, together with the measured area of the organelles, observed water permeability for intracellular CHO-K1 lysosomes was calculated to be 5.3 × 10⁻³ ± 0.3 × 10⁻³ cm/s. This work demonstrates the feasibility of measuring water flux into subcellular organelles in live cells using LY-dex.