Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy

Kaliszewski, Megan J.; Shi, Xiaojun; Hou, Yixuan J.; Lingerak, Ryan; Kim, Soyeon; Mallory, Paul; Smith, Adam W.

doi:10.1016/j.ymeth.2018.02.002

Cited by 41 publications

(57 citation statements)

References 57 publications

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“…The first option is a 1:1:2 Plexin D1-Nrp1-Semaphorin 3C complex. Here the median fc value falls within the range expected for simpledimerization, 49 but the values may be altered by monomeric Plexin D1 and dimeric Nrp1. If the Nrp1 homodimer has a high binding affinity it is also possible that Semaphorin 3C causes a 1:2:2 complex where a monomeric Plexin D1 binds to a Nrp1 dimer upon stimulation (Figure 8).…”

Section: Semaphorin 3c Induces Complex Formation For Plexin D1 and Nrsupporting

confidence: 62%

Resolving the Interactions between Class 3 Semaphorin Receptors in Live Cells

Christie

Hao

Tracy

et al. 2021

Preprint

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The plexin/neuropilin/semaphorin family of proteins is involved with tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion, but are also important in disease, including cancer angiogenesis and metastasis. While some structures of the soluble domains of these proteins have been determined, the conformations of full-length receptor complexes are just beginning to be studied, especially within the context of the cell plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) allows direct insight to the formation of protein-protein interactions in the membrane of live cells. Here we investigated the homodimerization of neuropilin-1, Plexin A2, Plexin A4, and Plexin D1. Consistent with previous studies, we found that neuropilin-1, Plexin A2 and Plexin A4 are dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that Plexin A2 and A4 assemble into a heteromeric complex. Stimulation with Semaphorin 3A or Semaphorin 3C ligand neither disrupts nor enhances the dimerization of the receptors when they are expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer-dimer equilibrium. However, upon stimulation with Semaphorin 3C, Plexin D1 and neuropilin-1 form a heteromeric complex, while Semaphorin 3A does not induce a stable complex with these receptors. This analysis of interactions by PIE-FCCS provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors during disease.

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Section: Semaphorin 3c Induces Complex Formation For Plexin D1 and Nrsupporting

confidence: 62%

Resolving the Interactions between Class 3 Semaphorin Receptors in Live Cells

Christie

Hao

Tracy

et al. 2021

Preprint

Self Cite

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“…3e , Supplementary Fig. 6b ), indicating that ligand stimulation induces dimerization and multimerization in both EGFR-WT and EGFR-KDD 36 , 38 . There is no statistically significant difference between EGFR-WT and EGFR-KDD, suggesting that the kinase duplication does not sterically restrict dimerization and multimerization.…”

Section: Resultsmentioning

confidence: 94%

Structure–function analysis of oncogenic EGFR Kinase Domain Duplication reveals insights into activation and a potential approach for therapeutic targeting

Brown

Kim

et al. 2021

Nat Commun

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Mechanistic understanding of oncogenic variants facilitates the development and optimization of treatment strategies. We recently identified in-frame, tandem duplication of EGFR exons 18 - 25, which causes EGFR Kinase Domain Duplication (EGFR-KDD). Here, we characterize the prevalence of ERBB family KDDs across multiple human cancers and evaluate the functional biochemistry of EGFR-KDD as it relates to pathogenesis and potential therapeutic intervention. We provide computational and experimental evidence that EGFR-KDD functions by forming asymmetric EGF-independent intra-molecular and EGF-dependent inter-molecular dimers. Time-resolved fluorescence microscopy and co-immunoprecipitation reveals EGFR-KDD can form ligand-dependent inter-molecular homo- and hetero-dimers/multimers. Furthermore, we show that inhibition of EGFR-KDD activity is maximally achieved by blocking both intra- and inter-molecular dimerization. Collectively, our findings define a previously unrecognized model of EGFR dimerization, providing important insights for the understanding of EGFR activation mechanisms and informing personalized treatment of patients with tumors harboring EGFR-KDD. Finally, we establish ERBB KDDs as recurrent oncogenic events in multiple cancers.

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“…between mEGFP and mEYFP for the polymerase subunits indicates higher order interactions, i.e. higher stoichiometry than 1:1 (37). To quantify the stoichiometry of the PC directly, we analyzed the molecular brightness of RSICS measurements for all three fluorophore species.…”

Section: Cross-correlation and Molecular Brightness Analysis Via Thrementioning

confidence: 99%

Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

Dunsing

Petrich

Chiantia

2020

Preprint

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Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify transport dynamics and interactions between biomolecules in living cells. For example, cross-correlation analysis of spectrally separated fluctuations allows the investigation of inter-molecular interactions. This analysis is conventionally limited to two fluorophore species that are excited with a single or two different laser lines and detected in two non-overlapping spectral channels. However, signaling pathways in biological systems often involve interactions between multiple biomolecules, e.g. formation of ternary or quaternary protein complexes. Here, we present a methodology to investigate such interactions at the plasma membrane (PM) of cells, as encountered for example in viral assembly or receptor-ligand interactions. To this aim, we introduce scanning fluorescence spectral correlation spectroscopy (SFSCS), a combination of scanning fluorescence correlation spectroscopy with spectrally resolved detection and decomposition. We first demonstrate that SFSCS allows cross-talk-free cross-correlation analysis of PM-associated proteins labeled with strongly overlapping fluorescent proteins (FPs), such as mEGFP and mEYFP, excited with a single excitation line. We then verify the applicability of SFSCS for quantifying diffusion dynamics and protein oligomerization (based on molecular brightness analysis) of two protein species tagged with spectrally overlapping FPs. Adding a second laser line, we demonstrate the possibility of three- and four-species (cross-) correlation analysis using mApple and mCherry2, as examples of strongly overlapping FP tags in the red spectral region. Next, we apply this scheme to investigate the interactions of influenza A virus (IAV) matrix protein 2 (M2) with two cellular host factors simultaneously. Using the same set of fluorophores, we furthermore extend the recently presented raster spectral image correlation spectroscopy (RSICS) approach to four species analysis, successfully demonstrating multiplexed RSICS measurements of protein interactions in the cell cytoplasm. Finally, we apply RSICS to investigate the assembly of the ternary IAV polymerase complex and report a 2:2:2 stoichiometry of these protein assemblies in the nucleus of living cells.

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Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy

Cited by 41 publications

References 57 publications

Resolving the Interactions between Class 3 Semaphorin Receptors in Live Cells

Resolving the Interactions between Class 3 Semaphorin Receptors in Live Cells

Structure–function analysis of oncogenic EGFR Kinase Domain Duplication reveals insights into activation and a potential approach for therapeutic targeting

Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

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