![]() These VLA-4 integrins have protein ligands of high affinity (e.g., fibronectin or VCAM-1 for α 4 integrin) and function to enable cell-to-cell and cell-to-extracellular matrix interactions, adhesion, and other behaviors important for immune response. Very late antigen-4 (VLA-4, α 4 β 1) is one class of integrins expressed on immune cells, such as leukocytes and antigen-presenting cells. Integrins are a family of transmembrane protein heterodimers that are expressed on the surface of different cell types. This work establishes a quantitative cytometric approach for profiling fluorescence donor decay kinetics during integrin conformational changes on a single-cell level. Last, a graphical phasor analysis, including population clustering, gating, and formation of an FRET trajectory, added precision to a comparative analysis of populations undergoing FRET, partial donor recovery, and complete donor recovery. Time-resolved flow cytometry analysis revealed donor emission increases up to 17%, fluorescence lifetime shifts over 1.0 ns during activation, and FRET efficiencies of 37% and 26% corresponding to the inactive and active integrin state, respectively. Integrin extension is determined by changes in the distance of closest approach between an FRET donor and acceptor, bound at the integrin head and cell membrane, respectively. The stalk-like transmembrane heterodimers when not active lay bent and upon activation extend outward. We exploit FRET to study the extent of activation of α4β1 integrin dimers expressed on the surface of leukocytes. ![]() When FRET as an optical technique is measured with flow cytometry, conformational changes of proteins can be rapidly measured cell-by-cell for the benefit of screening and profiling. Förster resonance energy transfer (FRET) continues to be a useful tool to study movement and interaction between proteins within living cells. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. Archives
February 2023
Categories |