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Indicator-dependent...
Indicator-dependent differences in detection of local intracellular Ca2+release events evoked by voltage-gated Ca2+entry in pancreatic & beta;-cells
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- Yang, Mingyu (author)
- Uppsala universitet,Institutionen för medicinsk cellbiologi
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- Dyachok, Oleg, 1965- (author)
- Uppsala universitet,Institutionen för medicinsk cellbiologi
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- Xu, Yunjian (author)
- Uppsala universitet,Institutionen för medicinsk cellbiologi
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- Gylfe, Erik, 1947- (author)
- Uppsala universitet,Institutionen för medicinsk cellbiologi
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- Idevall Hagren, Olof, 1980- (author)
- Uppsala universitet,Institutionen för medicinsk cellbiologi
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- Tengholm, Anders, 1971- (author)
- Uppsala universitet,Institutionen för medicinsk cellbiologi
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(creator_code:org_t)
- Elsevier BV, 2023
- 2023
- English.
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In: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 109
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https://uu.diva-port... (primary) (Raw object)
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Abstract
Subject headings
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- Genetically encoded Ca2+ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca2+ concentration ([Ca2+]i) changes in insulin-secreting & beta;-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K+ depolarizationtriggered submembrane [Ca2+]i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca2+]i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca2+]i spikes from an elevated level. [Ca2+]i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y1-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca2+ dyes. The indicator-dependent response patterns were unrelated to Ca2+ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca2+ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca2+ events originating outside this volume. The study highlights that voltage-dependent Ca2+ entry in & beta;-cells is tightly linked to local intracellular Ca2+ release mediated via an autocrine route that may be more important than previously reported direct Ca2+ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release.
Subject headings
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Fysiologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Physiology (hsv//eng)
Keyword
- Ca 2+oscillations
- R-GECO1
- Fluo-5F
- Fluo-4
- P2Y 1 receptor
- Insulin secretion
- TIRF microscopy
Publication and Content Type
- ref (subject category)
- art (subject category)
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