Using Fluorescent GAP Indicators to Monitor ER Ca 2 .

Autor: Rojo-Ruiz J; Unidad de Excelencia, Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain., Sánchez-Rabadán C; Unidad de Excelencia, Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain., Calvo B; Unidad de Excelencia, Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain., García-Sancho J; Unidad de Excelencia, Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain., Alonso MT; Unidad de Excelencia, Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Universidad de Valladolid y Consejo Superior de Investigaciones Científicas (CSIC), Valladolid, Spain.
Jazyk: angličtina
Zdroj: Current protocols [Curr Protoc] 2024 Jun; Vol. 4 (6), pp. e1060.
DOI: 10.1002/cpz1.1060
Abstrakt: The endoplasmic reticulum (ER) is the main reservoir of Ca 2+ of the cell. Accurate and quantitative measuring of Ca 2+ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca 2+ indicators have been developed, including a family of fluorescent Ca 2+ indicators, dubbed GFP-Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca 2+ -binding protein aequorin. GAP Ca 2+ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca 2+ concentrations; they are insensible to variations in the Mg 2+ concentrations or pH variations (in the 6.5-8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca 2+ affinity version of GAP, GAP3 (K D ≅ 489 µM), has been engineered to conform with the estimated [Ca 2+ ] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca 2+ . The ratiometric measurements provide a quantitative method to assess accurate [Ca 2+ ] ER , both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca 2+ indicators to simultaneously monitor ER and cytosolic Ca 2+ . Here, we provide detailed methods to assess erGAP3 expression and to perform Ca 2+ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescence Basic Protocol 2: Monitoring ER Ca 2+ Basic Protocol 3: Monitoring ER- and cytosolic-Ca 2+ Support Protocol: Generation of a stable cell line expressing erGAP3.
(© 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.)
Databáze: MEDLINE
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