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FRET-Based Nanobiosensors for Imaging Intracellular Ca²? and H? Microdomains.

ABSTRACT: Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H? transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) or H? probes. These 'Rubies' are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) for H? or Ca(2+) sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca(2+) and pH transients using live-cell fluorescence imaging.

SUBMITTER: Zamaleeva AI

PROVIDER: S-EPMC4610457 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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FRET-Based Nanobiosensors for Imaging Intracellular Ca²⁺ and H⁺ Microdomains.

Zamaleeva Alsu I AI Despras Guillaume G Luccardini Camilla C Collot Mayeul M de Waard Michel M Oheim Martin M Mallet Jean-Maurice JM Feltz Anne A

Sensors (Basel, Switzerland) 20150923 9

Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H⁺ transients. Our sensors combine a ...[more]

PMID: 26404317

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Project description:Intracellular calcium (Ca(2+)) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca(2+) binding proteins (Ca(2+) buffers) play an important role in regulating Ca(2+) concentration ([Ca(2+)]). Buffers typically slow [Ca(2+)] temporal dynamics and increase the effective volume of Ca(2+) domains. Because fluctuations in [Ca(2+)] decrease in proportion to the square-root of a domain's physical volume, one might conjecture that buffers decrease [Ca(2+)] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca(2+) signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca(2+) influx with passive exchange of both Ca(2+) and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca(2+) and buffer and use these stochastic differential equations to determine the magnitude of [Ca(2+)] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca(2+) signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca(2+)] fluctuations during periods of Ca(2+) influx, whereas stationary (immobile) Ca(2+) buffers do not. Also contrary to expectations, we find that in the absence of Ca(2+) influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca(2+)] fluctuations. We derive an analytical formula describing the influence of rapid Ca(2+) buffers on [Ca(2+)] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca(2+) buffers alter the dynamics of [Ca(2+)] fluctuations in a nonintuitive manner. The finding that Ca(2+) buffers do not suppress intrinsic domain [Ca(2+)] fluctuations raises the intriguing question of whether or not [Ca(2+)] fluctuations are a physiologically significant aspect of local Ca(2+) signaling.

Dataset Information

FRET-Based Nanobiosensors for Imaging Intracellular Ca²? and H? Microdomains.

Publications

FRET-Based Nanobiosensors for Imaging Intracellular Ca²⁺ and H⁺ Microdomains.

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