title: Cell-penetrating nanobiosensors for pointillistic intracellular Ca2+-transient detection.
abstract: Small-molecule chemical calcium (Ca(2+)) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca(2+) entry. Nanobiosensors incorporating functionalized quantum dots (QDs) have emerged as promising alternatives but their intracellular use remains a major challenge. We designed cell-penetrating FRET-based Ca(2+) nanobiosensors for the detection of local Ca(2+) concentration transients, using commercially available CANdot565QD as a donor and CaRuby, a custom red-emitting Ca(2+) indicator, as an acceptor. With Ca(2+)-binding affinities covering the range of 3-20 μM, our CaRubies allow building sensors with a scalable affinity for detecting intracellular Ca(2+) transients at various concentrations. To facilitate their cytoplasmic delivery, QDs were further functionalized with a small cell-penetrating peptide (CPP) derived from hadrucalcin (HadUF1-11: H11), a ryanodine receptor-directed scorpion toxin identified within the venom of Hadrurus gertschi. Efficient internalization of QDs doubly functionalized with PEG5-CaRuby and H11 (in a molar ratio of 1:10:10, respectively) is demonstrated. In BHK cells expressing a N-methyl-d-aspartate receptor (NMDAR) construct, these nanobiosensors report rapid intracellular near-membrane Ca(2+) transients following agonist application when imaged by TIRF microscopy. Our work presents the elaboration of cell-penetrating FRET-based nanobiosensors and validates their function for detection of intracellular Ca(2+) transients.
authors.affiliation: Ecole Normale Supérieure, Institut de Biologie de l'ENS (IBENS) , Paris F-75005, France.
authors.forename: Alsu I
authors.initials: AI
authors.lastname: Zamaleeva
authors.forename: Mayeul
authors.initials: M
authors.lastname: Collot
authors.forename: Eloi
authors.initials: E
authors.lastname: Bahembera
authors.forename: Céline
authors.initials: C
authors.lastname: Tisseyre
authors.forename: Philippe
authors.initials: P
authors.lastname: Rostaing
authors.forename: Aleksey V
authors.initials: AV
authors.lastname: Yakovlev
authors.forename: Martin
authors.initials: M
authors.lastname: Oheim
authors.forename: Michel
authors.initials: M
authors.lastname: de Waard
authors.forename: Jean-Maurice
authors.initials: JM
authors.lastname: Mallet
authors.forename: Anne
authors.initials: A
authors.lastname: Feltz
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D045663:Quantum Dots / Q000737:chemistry*; D012604:Scorpion Venoms / Q000737:chemistry
chemical_list: D057846:Cell-Penetrating Peptides; D012604:Scorpion Venoms; C544152:hadrucalcin protein, Hadrurus gertschi; D002118:Calcium