The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable tools. Here, we describe Voltair, a fluorescent DNA nanodevice that reports the ...absolute membrane potential and can be targeted to organelles in live cells. Voltair consists of a voltage-sensitive fluorophore and a reference fluorophore for ratiometry, and acts as an endocytic tracer. Using Voltair, we could measure the membrane potential of different organelles in situ in live cells. Voltair can potentially guide the rational design of biocompatible electronics and enhance our understanding of how membrane potential regulates organelle biology.
DNA Trojan horse: A DNA icosahedron (black, see scheme) held together with aptamers (red) was used to encapsulate molecular cargo such as fluorescent dextran (green). In the presence of a molecular ...trigger (gray hexagons), the aptamers fold back leading to opening of the icosahedron and simultaneous release of the encapsulated cargo.
The originally published paper has been updated to include the following new reference, added as ref. 18: Albrecht, T., Zhao, Y., Nguyen, T. H., Campbell, R. E. & Johnson, J. D. Fluorescent ...biosensors illuminate calcium levels within defined beta-cell endosome subpopulations. Cell Calcium 57, 263-274 (2015). Subsequent references have been renumbered in the reference list and throughout the text. Minor text changes were made in the sentence in which this new reference is first cited: "Previous attempts used endocytic tracers bearing either pH- or Ca
-sensitive dyes to serially measure population-averaged pH and apparent Ca
in different batches of cells, thus scrambling information from individual endosomes
" in the original introduction was changed to "Previous attempts used endocytic tracers bearing either pH- or Ca
-sensitive dyes
or fluorescent-protein-based sensors
to serially measure population-averaged pH and apparent Ca
in different batches of cells, thus scrambling information from individual endosomes." These changes have been made in the HTML and PDF versions of the article.
Lysosomes are multifunctional, subcellular organelles with roles in plasma membrane repair, autophagy, pathogen degradation and nutrient sensing. Dysfunctional lysosomes underlie Alzheimer's disease, ...Parkinson's disease and rare lysosomal storage diseases, but their contributions to these pathophysiologies are unclear. Live imaging has revealed lysosome subpopulations with different physical characteristics including dynamics, morphology or cellular localization. Here, we chemically resolve lysosome subpopulations using a DNA-based combination reporter that quantitatively images pH and chloride simultaneously in the same lysosome while retaining single-lysosome information in live cells. We call this technology two-ion measurement or 2-IM. 2-IM of lysosomes in primary skin fibroblasts derived from healthy individuals shows two main lysosome populations, one of which is absent in primary cells derived from patients with Niemann-Pick disease. When patient cells are treated with relevant therapeutics, the second population re-emerges. Chemically resolving lysosomes by 2-IM could enable decoding the mechanistic underpinnings of lysosomal diseases, monitoring disease progression or evaluating therapeutic efficacy.
Intracellular organelles are subsystems within a cell, whose activity and chemical composition reflect the metabolic state of live cells. Alterations in cellular homeostasis occurring in disease, ...ageing and development are also reflected at the level of organelles. By targeting organelles with pharmacological agents and genetic tools, the aim is to improve disease diagnosis and to restore cell function. In this Review, we discuss biological pathways that can be exploited to target the delivery of exogenous cargo with organelle-level precision. We investigate how these pathways can be leveraged for imaging, diagnosis and therapy at the organelle level, and highlight the potential of nucleic acids as delivery systems to target specific organelles in vivo, including the nucleus, lysosomes, secretory organelles and mitochondria. The programmability, modularity and biocompatibility of nucleic acid-based scaffolds make them well suited to accomplishing next-generation targeting with organelle-level resolution in living organisms.Organelles are compartments inside cells that play important parts in intercellular and intracellular communication, reflecting the metabolic state of the cell. This Review discusses biological pathways that can be exploited to target cargo to specific organelles in vivo, highlighting nucleic acids as suitable delivery vehicles for organelle-level imaging, diagnosis and therapy.
It is extremely challenging to quantitate lumenal Ca
in acidic Ca
stores of the cell because all Ca
indicators are pH sensitive, and Ca
transport is coupled to pH in acidic organelles. We have ...developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca
simultaneously, CalipHluor functions as a pH-correctable Ca
reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca
changes during endosomal maturation and found a surge in lumenal Ca
specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca
accumulation-an example of a lysosome-specific Ca
importer in animals. By enabling the facile quantification of compartmentalized Ca
, CalipHluor can expand the understanding of subcellular Ca
importers.
Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune ...cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca
gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.
A family of asymmetric thiazolo5,4-dthiazole (TTz) fluorescent dye sensors has been developed, and their photophysical sensing properties are reported. The π-conjugated, TTz-bridged compounds are ...synthesized via a single-step, double condensation/oxidation of dithiooxamide and two different aromatic aldehydes: one with strong electron-donating characteristics and one with strong electron-accepting characteristics. The four reported dyes include electron-donating moieties (N,N-dibutylaniline and N,N-diphenylaniline) matched with three different electron-accepting moieties (pyridine, benzoic acid, and carboxaldehyde). The asymmetric TTz derivatives exhibit strong solvatofluorochromism with Stokes shifts between 0.269 and 0.750 eV (2270 and 6050 cm–1) and transition dipole moments (Δμ = 13–18 D) that are among the highest reported for push–pull dyes. Fluorescence quantum yields are as high as 0.93 in nonpolar solvents, and the fluorescence lifetimes (τF) vary from 1.50 to 3.01 ns depending on the solvent polarity. In addition, thermofluorochromic studies and spectrophotometric acid titrations were performed and indicate the possibility of using these dyes as temperature and/or acid sensors. In vitro cell studies indicate good cell membrane localization, negligible cytotoxicity, promising voltage sensitivities, and photostabilities that are 4 times higher than comparable dyes. Their ease of synthesis and purification, remarkable photophysical properties, and chemically sensitive TTz π-bridge make these asymmetric dye derivatives attractive for environmental and biological sensing or similar molecular optoelectronic applications.
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