From fixed to FRAP: measuring protein mobility and activity in living cells

Reits, Eric; Neefjes, Jacques

doi:10.1038/35078615

Cited by 594 publications

(498 citation statements)

References 29 publications

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“…Fluorescence recovery was recorded over 5 min at 1 frame every 4 s. To control for movement in and out of focus, multiple regions of interest (ROI) were selected in nonphotobleached regions; only samples for which intensities within control ROIs remained steady throughout the FRAP experiment were used. Recovery data were further analyzed using Prism software (GraphPad, La Jolla, CA): mobile fraction was calculated as previously described (Reits and Neefjes, 2001 …”

Section: Fluorescence Recovery After Photobleaching (Frap)mentioning

confidence: 99%

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Pines

Fairchild

Tanentzapf

2010

Developmental Dynamics

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Cell adhesion must be precisely regulated to enable both dynamic morphogenetic processes and the subsequent transition to stable tissue maintenance. Integrins link the intracellular cytoskeleton and extracellular matrix, relaying bidirectional signals across the plasma membrane. In vitro studies have demonstrated that multiple mechanisms control integrin-mediated adhesion; however, their roles during development are poorly understood. We used mutations that activate or deactivate specific functions of vertebrate b-integrins in vitro to investigate how perturbing Drosophila bPS-integrin regulation in developing embryos affects tissue morphogenesis and maintenance. We found that morphogenetic processes use various b-integrin regulatory mechanisms to differing degrees and that conformational changes associated with outside-in activation are essential for developmental integrin functions. Long-term adhesion is also sensitive to integrin dysregulation, suggesting integrins must be continuously regulated to support stable tissue maintenance. Altogether, in vivo phenotypic analyses allowed us to identify the importance of various b-integrin regulatory mechanisms during different morphogenetic processes. Developmental Dynamics 240:36-51,

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Section: Fluorescence Recovery After Photobleaching (Frap)mentioning

confidence: 99%

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Pines

Fairchild

Tanentzapf

2010

Developmental Dynamics

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“…Several investigations have followed subcellular trafficking of MR and GR in living cells using green fluorescent protein (GFP) and its color variants (Htun et al, 1996;Fejes-Toth et al, 1998;Nishi et al, 2001;DeFranco, 2002). Furthermore, McNally et al (2000) showed that the hormone-occupied GR undergoes a rapid exchange between chromatin and the nucleoplasmic compartment by using the fluorescent recovery after photobleaching technique (Reits and Neefjes, 2001). We focused on the spatiotemporal-specific interactions between GR and MR in living cells.…”

Section: Introductionmentioning

confidence: 99%

Visualization of Glucocorticoid Receptor and Mineralocorticoid Receptor Interactions in Living Cells with GFP-Based Fluorescence Resonance Energy Transfer

Nishi¹,

Tanaka²,

Matsuda³

et al. 2004

J. Neurosci.

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Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the GR and MR are highly colocalized in the hippocampus. Given the differential action of the MR and GR in the hippocampal region, it is important to elucidate how these receptors interact with each other in response to corticosteroids. We investigated the heterodimerization of the MR and GR with green fluorescent protein-based fluorescence resonance energy transfer (FRET) microscopy in living cells with spatiotemporal manner. FRET was evaluated in three ways: (1) ratio imaging; (2) emission spectra; and (3) acceptor photobleaching. FRET analysis demonstrated that cyan fluorescent protein-GR and yellow fluorescent protein-MR form heterodimers after corticosterone (CORT) treatment both in the nucleus of cultured hippocampal neurons and COS-1 cells, whereas they do not form heterodimers in the cytoplasm. The content of the GR-MR heterodimer was higher at 10 Ϫ6 M CORT than at 10 Ϫ9 M CORT and reached a maximum level after 60 min of CORT treatment in both cultured hippocampal neurons and COS-1 cells. The distribution pattern of heterodimers in the nucleus of cultured hippocampal neurons was more restricted than that in COS-1 cells. The present study using mutant fusion proteins in nuclear localization signal showed that these corticosteroid receptors are not translocated into the nucleus in the form of heterodimers even after treatment with ligand and thus allow no heterodimerization to take place in the cytoplasm. These results obtained with FRET analyses give new insights into the sites, time course, and effects of ligand concentration on heterodimersization of the GR and MR.

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“…In bulk liquids, viscosity and diffusion constant are inversely proportional to each other. To investigate this relationship in two-dimensional membranes, and gain additional information on the effects of cholesterol, fluorescence recovery after photobleaching (FRAP) (Axelrod et al, 1976;Edidin et al, 1976;Reits and Neefjes, 2001) was used to measure the lateral diffusion of a lipid analog DiIC 12 .…”

Section: Introductionmentioning

confidence: 99%

The effect of cellular cholesterol on membrane-cytoskeleton adhesion

Sun

Northup

Marga

et al. 2007

Journal of Cell Science

170

161

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Whereas recent studies suggest that cholesterol plays important role in the regulation of membrane proteins, its effect on the interaction of the cell membrane with the underlying cytoskeleton is not well understood. Here, we investigated this by measuring the forces needed to extract nanotubes (tethers) from the plasma membrane, using atomic force microscopy. The magnitude of these forces provided a direct measure of cell stiffness, cell membrane effective surface viscosity and association with the underlying cytoskeleton. Furthermore, we measured the lateral diffusion constant of a lipid analog DiIC12, using fluorescence recovery after photobleaching, which offers additional information on the organization of the membrane. We found that cholesterol depletion significantly increased the adhesion energy between the membrane and the cytoskeleton and decreased the membrane diffusion constant. An increase in cellular cholesterol to a level higher than that in control cells led to a decrease in the adhesion energy and the membrane surface viscosity. Disassembly of the actin network abrogated all the observed effects, suggesting that cholesterol affects the mechanical properties of a cell through the underlying cytoskeleton. The results of these quantitative studies may help to better understand the biomechanical processes accompanying the development of atherosclerosis.

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From fixed to FRAP: measuring protein mobility and activity in living cells

Cited by 594 publications

References 29 publications

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Visualization of Glucocorticoid Receptor and Mineralocorticoid Receptor Interactions in Living Cells with GFP-Based Fluorescence Resonance Energy Transfer

The effect of cellular cholesterol on membrane-cytoskeleton adhesion

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