title: 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.
authors.affiliation: Department of Chemistry, Clemson University, Clemson, South Carolina 29634, USA.
authors.forename: Adriana
authors.initials: A
authors.lastname: Chaurra
authors.forename: Brittany M
authors.initials: BM
authors.lastname: Gutzman
authors.forename: Emily
authors.initials: E
authors.lastname: Taylor
authors.forename: P Christine
authors.initials: PC
authors.lastname: Ackroyd
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Christensen
mesh_terms: D000818:Animals; D016466:CHO Cells; D002463:Cell Membrane Permeability; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D017666:Deuterium Oxide / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D007546:Isoquinolines / Q000737:chemistry* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods; D012492:Salts / Q000378:metabolism; D014867:Water / Q000378:metabolism*
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D007546:Isoquinolines; D012492:Salts; D014867:Water; C017475:lucifer yellow; D017666:Deuterium Oxide