Membrane Mobility and Microdomain Association of the Dopamine Transporter Studied with Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching†

Biochemistry ◽  
2007 ◽  
Vol 46 (37) ◽  
pp. 10484-10497 ◽  
Author(s):  
Erika M. Adkins ◽  
Devadoss J. Samuvel ◽  
Jacob U. Fog ◽  
Jacob Eriksen ◽  
Lankupalle D. Jayanthi ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Correlation Spectroscopy ◽  
Fluorescence Recovery ◽  
Fluorescence Correlation

scholarly journals EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements

2019 ◽  
Vol 20 (13) ◽  
pp. 3370 ◽  
Author(s):  
György Vámosi ◽  
Elza Friedländer-Brock ◽  
Shehu M. Ibrahim ◽  
Roland Brock ◽  
János Szöllősi ◽  
...  
Keyword(s):  
Time Scales ◽  
Fluorescence Correlation Spectroscopy ◽  
Time Course ◽  
Fluorescence Recovery After Photobleaching ◽  
Slow Component ◽  
Chinese Hamster ◽  
Correlation Spectroscopy ◽  
Fluorescence Recovery ◽  
Brownian Diffusion ◽  
Fluorescence Correlation

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100–1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 μm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.

scholarly journals Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

2011 ◽  
Vol 22 (18) ◽  
pp. 3498-3507 ◽  
Author(s):  
Urszula Golebiewska ◽  
Jason G. Kay ◽  
Thomas Masters ◽  
Sergio Grinstein ◽  
Wonpil Im ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Surface Concentration ◽  
Correlation Spectroscopy ◽  
Similar Data ◽  
Fluorescence Correlation ◽  
The Many ◽  
Green Fluorescent

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

Investigating membrane protein dynamics in living cellsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

2006 ◽  
Vol 84 (6) ◽  
pp. 825-831 ◽  
Author(s):  
Ian R. Bates ◽  
Paul W. Wiseman ◽  
John W. Hanrahan
Keyword(s):  
Membrane Proteins ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Fluorescence Correlation ◽  
Transport Dynamics ◽  
Health And Disease ◽  
Image Correlation Spectroscopy ◽  
Selection Of

Live cell imaging is a powerful tool for understanding the function and regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based techniques for studying the transport dynamics of membrane proteins: fluorescence-correlation spectroscopy, image-correlation spectroscopy, fluorescence recovery after photobleaching, and single-particle and (or) molecule tracking. The advantages and limitations of each approach are illustrated using recent studies of an ion channel and cell adhesion molecules.

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