44 research outputs found
Small molecule anionophores promote transmembrane anion permeation matching CFTR activity
Anion selective ionophores, anionophores, are small molecules capable of facilitating the
transmembrane transport of anions. Inspired in the structure of natural product prodigiosin, four
novel anionophores 1a-d, including a 1,2,3-triazole group, were prepared. These compounds proved
highly efficient anion exchangers in model phospholipid liposomes. The changes in the hydrogen bond
cleft modified the anion transport selectivity exhibited by these compounds compared to prodigiosin
and suppressed the characteristic high toxicity of the natural product. Their activity as anionophores
in living cells was studied and chloride efflux and iodine influx from living cells mediated by these
derivatives was demonstrated. These compounds were shown to permeabilize cellular membranes
to halides with efficiencies close to the natural anion channel CFTR at doses that do not compromise
cellular viability. Remarkably, optimal transport efficiency was measured in the presence of pH
gradients mimicking those found in the airway epithelia of Cystic Fibrosis patients. These results
support the viability of developing small molecule anionophores as anion channel protein surrogates
with potential applications in the treatment of conditions such as Cystic Fibrosis derived from the
malfunction of natural anion transport mechanisms.European Union’s Horizon 2020 research and innovation programme under grant agreement No. 667079, La Marató de TV3 Foundation (20132730), Consejería de Educación de la Junta de Castilla y León (Projects BU340U13 and BU092U16
Imaging of endosome fusion in BHK fibroblasts based on a novel fluorimetric avidin-biotin binding assay
A fluorescence assay of in vivo endosome fusion was developed and applied to define the kinetics of endosome fusion in baby hamster kidney (BHK) fibroblasts. The assay is based on an approximately 10-fold enhancement of the green fluorescence of BODIPY-avidin upon biotin binding. The BODIPY-avidin fluorescence enhancement occurred in < 25 ms, was pH-independent, and involved a BODIPY-tryptophan interaction. For endocytosis in vivo, BHK fibroblasts were pulse-labeled with BODIPY-avidin together with a red (rhodamine) fluorescent fusion-independent chromophore (TMR). After specified chase times in a nonfluorescent medium, a second cohort of endosomes was pulse-labeled with biotin-conjugated albumin, dextran, or transferrin. Fusion of biotin-containing endosomes with avidin-containing endosomes was quantified by ratio imaging of BODIPY-to-TMR fluorescence in individual endosomes, using imaging methods developed for endosome pH studies. Analysis of BODIPY-to-TMR ratio distributions in avidin-labeled endosomes exposed to zero and maximum biotin indicated > 90% sensitivity for detection of endosome fusion. In avidin pulse (10 min) -chase-biotin albumin pulse (10 min) studies, both fused and unfused endosomes were identified; the fractions of avidin-labeled endosomes that fused with biotin-labeled endosomes were 0.48, 0.21, 0.16, and 0.07 for 0-, 5-, 10-, and 20-min chase times. Fitting of fusion data to a mathematical model of in vivo endosome fusion required the existence of an intermediate fusion compartment. Pulse-chase studies performed with biotin-transferrin to label the early/recycling endosomes indicated that after a 10-min chase, avidin-labeled endosomes reached a compartment that was inaccessible to biotin-transferrin. The assay was also applied to determine whether endosome fusion was influenced by temperature, pH (bafilomycin A1), second messengers (cAMP agonists, phorbol 12-myristate 13-acetate, staurosporine), and growth-related factors (platelet-derived growth factor, genistein). The results establish a sensitive fluorescence assay to quantify the fusion of vesicular compartments in living cells
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Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts.
Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers