Abstract：
:The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion) to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE's success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE's direct measurement and ease of use make it appealing for cell biologists.
journal_name
Cell Repjournal_title
Cell reportsauthors
Gura Sadovsky R,Brielle S,Kaganovich D,England JLdoi
10.1016/j.celrep.2017.02.063subject
Has Abstractpub_date
2017-03-14 00:00:00pages
2795-2806issue
11issn
2211-1247pii
S2211-1247(17)30278-4journal_volume
18pub_type
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