Direct measurement of Gag–Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy

Larson, Daniel R.; May, Yu; Vogt, Volker M.; Webb, Watt W.

doi:10.1083/jcb.200303200

Cited by 104 publications

(114 citation statements)

References 53 publications

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“…To exclude alternative interpretations of the results, numerous controls must be performed and the fluorescence intensities must be measured with high quantitative accuracy. Despite these limitations, FRET has been successfully used for the analysis of many protein interactions in living cells (Sorkin, et al, 2000, Li, et al, 2001, Majoul, et al, 2002, Hink, et al, 2003, Larson, et al, 2003, Miyawaki, 2003, Tsien, 2003. A great advantage of FRET relative to BiFC analysis is the real-time detection of complex formation and dissociation, which potentially allows analysis of the interaction under equilibrium conditions.…”

Section: Ubiquitin Family Peptide Conjugates That Have Been Visualizementioning

confidence: 99%

Bimolecular Fluorescence Complementation: Visualization of Molecular Interactions in Living Cells

Kerppola

2008

Methods in Cell Biology

130

109

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A variety of experimental methods have been developed for the analysis of protein interactions. The majority of these methods either require disruption of the cells to detect molecular interactions or rely on indirect detection of the protein interaction. The bimolecular fluorescence complementation (BiFC) assay provides a direct approach for the visualization of molecular interactions in living cells and organisms. The BiFC approach is based on the facilitated association between two fragments of a fluorescent protein when the fragments are brought together by an interaction between proteins fused to the fragments. The BiFC approach has been used for visualization of interactions among a variety of structurally divers interaction partners in many different cell types and. It enables detection of transient complexes as well as complexes formed by a subpopulation of the interaction partners. It is essential to include negative controls in each experiment in which the interface between the interaction partners has been mutated or deleted. The BiFC assay has been adapted for simultaneous visualization of multiple protein complexes in the same cell and the competition for shared interaction partners. A ubiquitin-mediated fluorescence complementation (UbFC) assay has also been developed for visualization of the covalent modification of proteins by ubiquitin family peptides. These fluorescence complementation assays have a great potential to illuminate a variety of biological interactions in the future.

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Section: Ubiquitin Family Peptide Conjugates That Have Been Visualizementioning

confidence: 99%

Bimolecular Fluorescence Complementation: Visualization of Molecular Interactions in Living Cells

Kerppola

2008

Methods in Cell Biology

130

109

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“…Indeed, FRET has been demonstrated between retroviral Gag-CFP and Gag-YFP molecules in whole cells [16][17][18] . To monitor the assembly of individual particles we chose the comparatively weak FRET pair of GFP/mCherry 19 , precisely because their absorption and emission spectra overlap less than those of CFP and YFP.…”

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confidence: 99%

Imaging the biogenesis of individual HIV-1 virions in live cells

Jouvenet

Bieniasz

Simon

2008

Nature

299

375

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Observations of individual virions in live cells have led to the characterization of their attachment, entry and intracellular transport 1 . However, the assembly of individual virions has never been observed in real-time. Insights into this process have come primarily from biochemical analyses of populations of virions or from microscopic studies of fixed infected cells. Thus, some assembly properties, such as kinetics and location, are either unknown or controversial 2-5 . Here, we quantitatively describe the genesis of individual virions in real-time, from initiation of assembly to budding and release. We studied fluorescently tagged derivatives of Gag, the major structural component of HIV-1 which is sufficient to drive assembly of viral-like-particles (VLPs) 6 , using fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescent microscopy (TIR-FM) in living cells. Virions appeared individually at the plasma membrane, their assembly rate accelerated as Gag protein accumulated in cells and typically five to six minutes was required to complete the assembly of a single virion. These approaches allow a previously unobserved view of the genesis of individual virions and the determination of parameters of viral assembly that are inaccessible using conventional techniques.We monitored HeLa cells coexpressing untagged Gag and Gag fused to green fluorescent protein (GFP). This avoids the reported morphologic defect seen when Gag-GFP is expressed alone 7 and electron microscopic studies confirmed that VLPs containing Gag and Gag-GFP were morphologically indistinguishable from VLPs containing Gag only (data not shown). Gag was detected as a diffuse signal at 5-6 hours after transfection and discrete puncta that apparently localized at the plasma membrane began to appear at 6-7 after transfection ( Supplementary Fig. 1a) 4 . However, the signal from puncta at the plasma membrane was partially masked by the strong diffuse fluorescence emitted by cytoplasmic Gag-GFP ( Supplementary Fig. 1b). This diffuse fluorescence diminished relative to the intensity of the fluorescent puncta when imaged with TIR-FM, whose illumination decays exponentially from the coverslip/medium interface with a space constant of ~70 nm 8 , (Supplementary Fig. 1b). Thus, TIR-FM is well suited to monitor appearance of VLPs at the plasma membrane.Cells that had generated few (less than 20) Gag puncta at 5 to 6 after transfection were observed over the ensuing 30 to 60 min ( Supplementary Fig. 1c). Typically, 50 to 150 puncta per cell appeared during this period (movie 1). Their behavior fell into two discrete classes. One class appeared over several minutes (Fig. 1a top, b left and movie 2) and showed little lateral movement during and after emergence (average lateral velocity = 0.007 µm/sec, n=25,

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“…6). Measurements of intracellular protein dynamics have made important insights into retroviral assembly (Larson et al, 2003), intracellular motility (Kohler et al, 2000), cytoplasmic structure (Weiss et al, 2003), and cell cycle control (Wang et al, 2006). On-going developments in fluorescent proteins and fluorophores (Kogure et al, 2006) and data analysis methods (Chen et al, 1999Muller, 2004) promise to provide new insights into cellular dynamics.…”

Section: Cellular Applicationsmentioning

confidence: 99%