A genome-wide functional assay of signal transduction in living mammalian cells

Whitney, Mike; Rockenstein, Edward; Cantin, Greg T.; Knapp, Tom; Zlokarnik, Gregor; Sanders, Pam; Durick, Kyle; Craig, F F; Negulescu, Paul A.

doi:10.1038/4302

Cited by 68 publications

(45 citation statements)

References 27 publications

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“…We set out to create a quantitative cell-cell fusion assay that (i) could be measured in real time (i.e., did not require induction of gene expression), (ii) could be done with a 96-well plate, (iii) did not require complicated image processing, and (iv) could be measured using a standard multiwell plate reader (e.g., luminometer, spectrophotometer, or fluorometer). Our solution to this problem was to measure changes in fluorescence generated after mixing two cell populations, one of which was loaded with a reporter fluorophore ( (32,38). When excited at 405 nm, intact CCF2 displays bright green fluorescence, whereas the ␤-lactamase cleavage product displays bright blue fluorescence.…”

Section: Resultsmentioning

confidence: 99%

Altering Expression Levels of Human Immunodeficiency Virus Type 1 gp120-gp41 Affects Efficiency but Not Kinetics of Cell-Cell Fusion

Lineberger

Danzeisen

Hazuda

et al. 2002

J Virol

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Human immunodeficiency virus (HIV) entry into a host cell requires the fusion of virus and cellular membranes that is driven by interaction of the viral envelope glycoproteins gp120 and gp41 (gp120/gp41) with CD4 and a coreceptor, typically either CXCR4 or CCR5. The stoichiometry of gp120/gp41:CD4:CCR5 necessary to initiate membrane fusion is not known. To allow an examination of early events in gp120/gp41-driven membrane fusion, we developed a novel real-time cell-cell fusion assay. Using this assay to study fusion kinetics, we found that altering the cell surface density of gp120/gp41 affected the maximal extent of fusion without dramatically altering fusion kinetics. Collectively, these observations are consistent with the view that gp120/gp41-driven membrane fusion requires the formation of a threshold number of fusion-active intercellular gp120/gp41:CD4:CCR5 complexes. Furthermore, the probability of reaching this threshold is governed, in part, by the surface density of gp120/gp41.Though much progress has been made toward understanding the mechanism whereby human immunodeficiency virus (HIV) gp120 and gp41 (gp120/gp41) binding to CD4 and appropriate coreceptors triggers virus entry, many questions remain. One question of central importance is, what stoichiometry of gp120/gp41 trimers, CD4 molecules, and coreceptor molecules (gp120/gp41:CD4:CoR complex) is needed to initiate membrane fusion? The efficiency of forming fusogenic gp120/gp41:CD4:CoR complexes, regardless of the precise stoichiometry that must be achieved, likely depends on many factors, such as the cell (or virion) surface densities of the relevant molecules (24), their quaternary structure (e.g., coreceptor dimerization) (30) or preexisting CD4-coreceptor interactions (15,17,37), and possibly the conformational state or microenvironment of the coreceptor (16).Kabat and coworkers have proposed a mass-action model to explain their findings that a threshold density of CCR5 is needed to support HIV infection and that the threshold level of CCR5 varies according to CD4 expression (14,24). An extension of the mass-action model of complex formation is that the virion-surface density (or cell-surface density) of gp120/gp41 is likely to be an important determinant of how efficiently fusion-competent gp120/gp41:CD4:CoR complexes form. Understanding of the fusion mechanism of influenza virus hemagglutinin (HA) protein has benefited greatly from studies in which fusogenic activity has been correlated with HA expression levels (5, 8). In principle, similar studies applied to HIV gp120/gp41-driven membrane fusion might reveal the minimum number of gp120/gp41 molecules necessary to promote membrane fusion. To date, this parameter has not been investigated systematically, in part because of a dearth of experimental systems in which to study early gp120/gp41 function. Here we describe the use of a new cell-cell fusion assay capable of monitoring gp120/gp41 function on a short time scale (minutes to hours) to address this issue. MATERIALS AND METHODS Cells. Sup...

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Section: Resultsmentioning

confidence: 99%

Altering Expression Levels of Human Immunodeficiency Virus Type 1 gp120-gp41 Affects Efficiency but Not Kinetics of Cell-Cell Fusion

Lineberger

Danzeisen

Hazuda

et al. 2002

J Virol

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“…for both positive and negative selection by FACS (61)(62)(63)(64). One would stain and sort for bla expression [blue fluorescence with coumarin-cephalosporin-fluorescein 2/acetoxymethyl ester (CCF2/ AM) live-cell substrate] 1 h or so after exposure to red light and no bla expression (green fluorescence with CCF2/AM) after near-IR or no light.…”

Section: Apply Snapshot Reporter To Mark Cells Activated During Amygdmentioning

confidence: 99%

Very long-term memories may be stored in the pattern of holes in the perineuronal net

Tsien

2013

Proc. Natl. Acad. Sci. U.S.A.

210

189

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A hypothesis and the experiments to test it propose that very long-term memories, such as fear conditioning, are stored as the pattern of holes in the perineuronal net (PNN), a specialized ECM that envelops mature neurons and restricts synapse formation. The 3D intertwining of PNN and synapses would be imaged by serial-section EM. Lifetimes of PNN vs. intrasynaptic components would be compared with pulse-chase 15 N labeling in mice and 14 C content in human cadaver brains. Genetically encoded indicators and antineoepitope antibodies should improve spatial and temporal resolution of the in vivo activity of proteases that locally erode PNN. Further techniques suggested include genetic KOs, better pharmacological inhibitors, and a genetically encoded snapshot reporter, which will capture the pattern of activity throughout a large ensemble of neurons at a time precisely defined by the triggering illumination, drive expression of effector genes to mark those cells, and allow selective excitation, inhibition, or ablation to test their functional importance. The snapshot reporter should enable more precise inhibition or potentiation of PNN erosion to compare with behavioral consequences. Finally, biosynthesis of PNN components and proteases would be imaged.

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“…The 29-kDa TEM-1 isoform of Bla has become a popular ''reporter'' or ''sensor'' of biological processes and interactions, such as the activity of promoter͞regulatory elements in living mammalian tissue culture cells (10,11), constitutive and inducible protein associations (12)(13)(14), and HIV-1 virion fusion in primary T lymphocytes (15). Bla is relatively small, monomeric, and naturally absent from eukaryotes, but it is easily expressible in eukaryotic cells without noticeable toxicity.…”

mentioning

confidence: 99%