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 D(2)O sensitive dye, and a D(2)O insensitive control dye, Alexa fluor 546 dextran (AF546-dex). LY-dex responded linearly to changes in D(2)O concentration and the LY-dex D(2)O 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 D(2)O 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 D(2)O. 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.

Current methods to analyze lysosome morphology, positioning, motility and function

“…Dextrans are biologically inert because they are composed of a poly‐(α‐ d ‐1,6‐glucose) linkage that cannot be degraded by lysosomal hydrolases. Dextrans are available linked to different fluorophores such as Alexa Fluor, Oregon Green and Lucifer Yellow, the latter being useful also to monitor water flux in intracellular organelles and lysosomes given its sensitivity to deuterium (D 2 O) concentration 72 . Fluorescence intensity of lysosomal‐localized Lucifer Yellow‐dextran increases in cells incubated in D 2 O‐containing medium according to the extent of D 2 O influx in lysosomes.…”

LRRC8 family proteins within lysosomes regulate cellular osmoregulation and enhance cell survival to multiple physiological stresses

“…A fluorescence assay was used to investigate water flow across the lysosomal membrane. Lysosomes within intact cells were loaded with Lucifer yellow dextran (LY-Dextran), which displays enhanced fluorescence in the presence of deuterated water (D 2 O) (40). Hypoosmotic challenge with D 2 O quickly augmented the fluorescence intensity (Fig.…”

Red-Emitting Fluorophores as Local Water-Sensing Probes

“…Yet, quantitative fluorescent probes that can report on changes in local water contents in the different crowded and heterogeneous environments of a cell are scarce. Existing emissive probes can detect trace amounts of water in organic solvents or water transport in organelles, but they are based on ratiometric detection or are incompatible either with high water contents and thereby with a cellular environment or with single-molecule detection because of a poor absorption coefficient and excitation below 500 nm. − Furthermore, although many fluorophores exist for single-molecule and super-resolution imaging, very few have been used as environmental reporters at the single-molecule level. ,− …”