Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques.

Published

Journal Article

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the Förster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Full Text

Duke Authors

Cited Authors

  • Rothenberg, KE; Puranam, I; Hoffman, BD

Published Date

  • November 2, 2018

Published In

PubMed ID

  • 30451225

Pubmed Central ID

  • 30451225

Electronic International Standard Serial Number (EISSN)

  • 1940-087X

International Standard Serial Number (ISSN)

  • 1940-087X

Digital Object Identifier (DOI)

  • 10.3791/58619

Language

  • eng