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CaBLAM! A high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase.
Lambert, Gerard G; Crespo, Emmanuel L; Murphy, Jeremy; Boassa, Daniela; Luong, Selena; Celinskis, Dmitrijs; Venn, Stephanie; Hu, Junru; Sprecher, Brittany; Tree, Maya O; Orcutt, Richard; Heydari, Daniel; Bell, Aidan B; Torreblanca-Zanca, Albertina; Hakimi, Ali; Lipscombe, Diane; Moore, Christopher I; Hochgeschwender, Ute; Shaner, Nathan C.
Afiliación
  • Lambert GG; Department of Neurosciences, University of California San Diego School of Medicine, La Jolla, CA USA.
  • Crespo EL; College of Medicine, Central Michigan University, Mt. Pleasant, MI USA.
  • Murphy J; Carney Institute for Brain Sciences, Department of Neuroscience, Brown University, Providence, RI USA.
  • Boassa D; Department of Neurosciences, University of California San Diego School of Medicine, La Jolla, CA USA.
  • Luong S; University of California San Diego, La Jolla, CA USA.
  • Celinskis D; Carney Institute for Brain Sciences, Department of Neuroscience, Brown University, Providence, RI USA.
  • Venn S; College of Medicine, Central Michigan University, Mt. Pleasant, MI USA.
  • Hu J; National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA USA.
  • Sprecher B; Department of Neurosciences, University of California San Diego School of Medicine, La Jolla, CA USA.
  • Tree MO; College of Medicine, Central Michigan University, Mt. Pleasant, MI USA.
  • Orcutt R; Department of Neurosciences, University of California San Diego School of Medicine, La Jolla, CA USA.
  • Heydari D; Department of Neurosciences, University of California San Diego School of Medicine, La Jolla, CA USA.
  • Bell AB; University of California San Diego, La Jolla, CA USA.
  • Torreblanca-Zanca A; Department of Neurosciences, University of California San Diego School of Medicine, La Jolla, CA USA.
  • Hakimi A; Scintillon Institute, San Diego, CA USA.
  • Lipscombe D; College of Medicine, Central Michigan University, Mt. Pleasant, MI USA.
  • Moore CI; Carney Institute for Brain Sciences, Department of Neuroscience, Brown University, Providence, RI USA.
  • Hochgeschwender U; College of Medicine, Central Michigan University, Mt. Pleasant, MI USA.
  • Shaner NC; Department of Neurosciences, University of California San Diego School of Medicine, La Jolla, CA USA.
bioRxiv ; 2023 Oct 04.
Article en En | MEDLINE | ID: mdl-37425712
ABSTRACT
Ca2+ plays many critical roles in cell physiology and biochemistry, leading researchers to develop a number of fluorescent small molecule dyes and genetically encodable probes that optically report changes in Ca2+ concentrations in living cells. Though such fluorescence-based genetically encoded Ca2+ indicators (GECIs) have become a mainstay of modern Ca2+ sensing and imaging, bioluminescence-based GECIs-probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein-have several distinct advantages over their fluorescent counterparts. Bioluminescent tags do not photobleach, do not suffer from nonspecific autofluorescent background, and do not lead to phototoxicity since they do not require the extremely bright extrinsic excitation light typically required for fluorescence imaging, especially with 2-photon microscopy. Current BL GECIs perform poorly relative to fluorescent GECIs, producing small changes in bioluminescence intensity due to high baseline signal at resting Ca2+ concentrations and suboptimal Ca2+ affinities. Here, we describe the development of a new bioluminescent GECI, "CaBLAM," which displays a much higher contrast (dynamic range) than previously described bioluminescent GECIs coupled with a Ca2+ affinity suitable for capturing physiological changes in cytosolic Ca2+ concentration. Derived from a new variant of Oplophorus gracilirostris luciferase with superior in vitro properties and a highly favorable scaffold for insertion of sensor domains, CaBLAM allows for single-cell and subcellular resolution imaging of Ca2+ dynamics at high frame rates in cultured neurons. CaBLAM marks a significant milestone in the GECI timeline, enabling Ca2+ recordings with high spatial and temporal resolution without perturbing cells with intense excitation light.

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: BioRxiv Año: 2023 Tipo del documento: Article Pais de publicación: EEUU / ESTADOS UNIDOS / ESTADOS UNIDOS DA AMERICA / EUA / UNITED STATES / UNITED STATES OF AMERICA / US / USA

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: BioRxiv Año: 2023 Tipo del documento: Article Pais de publicación: EEUU / ESTADOS UNIDOS / ESTADOS UNIDOS DA AMERICA / EUA / UNITED STATES / UNITED STATES OF AMERICA / US / USA