Investigating The Landscape Of Intracellular [Ca2+] In Live Cells By Rapid Photoactivated Cross-Linking Of Calmodulin-Protein Interactions
CELL CALCIUM(2021)
摘要
The Ca2+ sensor protein calmodulin interacts in a Ca2+-dependent manner with a large number of proteins that among them encompass a diverse assortment of functions and subcellular localizations. A method for monitoring calmodulin-protein interactions as they occur throughout a living cell would thus uniquely enable investigations of the intracellular landscape of [Ca2+] and its relationship to cell function. We have developed such a method based on capture of calmodulin-protein interactions by rapid photoactivated cross-linking (t1/2 -7s) in cells stably expressing a tandem affinity tagged calmodulin that have been metabolically labeled with a photoreactive methionine analog. Tagged adducts are stringently enriched, and captured calmodulin interactors are then identified and quantified based on tandem mass spectrometry data for their tryptic peptides. In this paper we show that the capture behaviors of interactors in cells are consistent with the presence of basal microdomains of elevated [Ca2+]. Ca2+ sensitivities for capture were determined, and these suggest that [Ca2+] levels are above -1 mu M in these regions. Although the microdomains appear to affect capture of most proteins, capture of some is at an apparent Ca2+-dependent maximum, suggesting they are targeted to the domains. Removal of extracellular Ca2+ has both immediate (5 min) and delayed (30 min) effects on capture, implying that the microdomains are supported by a combination of Ca2+ influx across the cell membrane and Ca2+ derived from internal stores. The known properties of the presumptive microdomain targeted proteins suggestroles in a variety of Ca2+-dependent basal metabolism and in formation and maintenance of the domains.
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关键词
Calmodulin, Calcium, Cross-linking, Microdomains, Interactome
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