Abstract
The dynamic regulation of signal transduction at plasma membrane microdomains remains poorly understood due to limitations in current experimental approaches. Genetically encoded biosensors based on fluorescent resonance energy transfer (FRET) can provide high spatiotemporal resolution for imaging cell signaling networks. Here, distinctive regulation of focal adhesion kinase (FAK) and Ca2+ signals are visualized at different membrane microdomains by FRET using membrane‐targeting biosensors. It is shown that rigidity‐dependent FAK and Ca2+ signals in human mesenchymal stem cells (hMSCs) are selectively activated at detergent‐resistant membrane (DRM or rafts) microdomains during the cell–matrix adhesion process, with minimal activities at non‐DRM domains. The rigidity‐dependent Ca2+ signal at the DRM microdomains is downregulated by either FAK inhibition or lipid raft disruption, suggesting that FAK and lipid raft integrity mediate the in situ Ca2+ activation. It is further revealed that transient receptor potential subfamily M7 (TRPM7) participates in the mobilization of Ca2+ signals within DRM regions. Thus, the findings provide insights into the underlying mechanisms that regulate Ca2+ and FAK signals in hMSCs under different mechanical microenvironments.
Highlights
Cell-based therapeutics are revolutionizing the medicine field.[1]
We developed and utilized two kinds of membrane-targeting focal adhesion kinase (FAK) biosensors based on fluorescent resonance energy transfer (FRET) technology
A detergent-resistant membrane (DRM)-targeting FAK biosensor, called Lyn-FAK, was engineered containing a lipid raft-targeting motif (MGCIKSKRKDNLNDDE) originated from Lyn kinase to the N-terminus of cytosolic FAK, which enables the tethering of this sensor to the DRM microdomain
Summary
Cell-based therapeutics are revolutionizing the medicine field.[1]. One promising branch is stem cell-based therapy, which has developed from preclinical to early clinical studies for treatment of various diseases.[2]. Y. Wang Department of Bioengineering University of Illinois at Urbana-Champaign Urbana, IL 61801, USA owing to the differential distribution of membrane-associated proteins at plasma membrane microdomains. Wang Department of Bioengineering University of Illinois at Urbana-Champaign Urbana, IL 61801, USA owing to the differential distribution of membrane-associated proteins at plasma membrane microdomains It remains unclear how signaling events are compartmentalized by specialized microdomains due to limitations in available methodologies. We take advantage of membrane-targeting fluorescent resonance energy transfer (FRET)-based FAK and Ca2+ biosensors to investigate the regulation of these two signals during cell–matrix adhesion process in hMSCs seeded on substrates with different stiffness. Through live single cell imaging, we first demonstrate that rigidity-dependent FAK and Ca2+ signals are selectively enriched at detergent-resistant membrane (DRM) microdomains in a concerted manner, but not at non-DRM during this adhesion process. We report that transient receptor potential subfamily M7 (TRPM7) is involved in the regulation of matrix rigidity-dependent Ca2+ signals at DRM microdomains, which is mediated by the functional FAK and the integrity of lipid rafts
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