An improved confocal FRAP technique for the measurement of long-term actin dynamics in individual stress fibers.
The present study describes an improved fluorescent recovery after photobleaching (FRAP) technique, which has been successfully used to quantify actin dynamics within individual fibers. Chondrocytes were transfected with an eGFP-actin plasmid and cultured on glass coverslips. In cells expressing eGFP-actin, confocal microscopy was used to bleach 3 x 1 microm regions accurately positioned along individual stress fibers. The subsequent fluorescent recovery over a 10-min imaging period was assessed from a series of intensity profiles, positioned along the length of the stress fibers and spanning the bleach region. From these profiles, the normalized fluorescent intensity values were plotted against time. In this way, the technique provided sufficient spatial precision to describe the long-term behavior within individual stress fibers while accounting for the inherent movement. An identical procedure was used to examine FRAP for eGFP-actin within the interfiber region. The FRAP curves for stress fibers were accurately modeled by two phase exponentials which indicated only partial recovery with a mobile fraction of 46%. This suggests that some of the F-actin molecules were in a tightly bound configuration with negligible turnover. The interfiber region exhibited similar two phase exponential FRAP with a mobile fraction of 68%. This partial recovery may be due to the presence, within the interfiber region, of both G-actin and fine F-actin fibers beneath the resolution of the confocal microscope. In conclusion, the present FRAP methodology overcomes many of the limitations of previous studies in order to provide new data describing long-term actin dynamics within individual stress fibers.
Campbell
J J
JJ
Department of Engineering, Medical Engineering Division, Queen Mary University of London, London E1 4NS, United Kingdom.
Knight
M M
MM
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002478:Cells, Cultured; D005453:Fluorescence; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D022502:Stress Fibers / Q000378:metabolism / Q000648:ultrastructure*
D000199:Actins; D049452:Green Fluorescent Proteins