One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

Fricke, Franziska; Beaudouin, Joël; Eils, Roland; Heilemann, Mike

doi:10.1038/srep14072

Cited by 160 publications

(226 citation statements)

References 54 publications

(64 reference statements)

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“…As shown in Figure 3C, for CD80 fused with mEos2 and expressed in HeLa cells (Fricke et al. , 2015a), neither the monomer nor the dimer alone can explain the observed blinking statistics.…”

Section: Resultsmentioning

confidence: 98%

“…Figure 3A compares the observed blinking statistics for CD86 monomers fused to mEos2 (Fricke et al. , 2015a) to the predictions for m = 0, 1, and 2 with fixed p = 0.289 and q = 0.295.…”

Section: Resultsmentioning

confidence: 99%

“…Figure 3B compares the blinking statistics observed for trimers of vesicular stomatitis virus glycoprotein (VSVG) fused to mEos2 (Fricke et al. , 2015a) to the predictions up to tetramers, with fixed p = 0.289 and q = 0.295.…”

Section: Resultsmentioning

confidence: 99%

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Model-independent counting of molecules in single-molecule localization microscopy

Hummer

Fricke

Heilemann

2016

MBoC

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147

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“…As shown in Figure 3C, for CD80 fused with mEos2 and expressed in HeLa cells (Fricke et al. , 2015a), neither the monomer nor the dimer alone can explain the observed blinking statistics.…”

Section: Resultsmentioning

confidence: 98%

“…Figure 3A compares the observed blinking statistics for CD86 monomers fused to mEos2 (Fricke et al. , 2015a) to the predictions for m = 0, 1, and 2 with fixed p = 0.289 and q = 0.295.…”

Section: Resultsmentioning

confidence: 99%

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Model-independent counting of molecules in single-molecule localization microscopy

Hummer

Fricke

Heilemann

2016

MBoC

Self Cite

147

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“…As an additional control, we also analyzed the membrane organization of a single-pass transmembrane protein, CD86 [previously demonstrated to be monomeric (37)], tagged at the C terminus with mEos, and observed a similar peak in the paircorrelation function (Fig. S2E).…”

Section: When Expressed Alone Egfr But Not Her3 Clusters In Responmentioning

confidence: 99%

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms

Lengerich

Agnew

Puchner

et al. 2017

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

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Heteromeric interactions between the catalytically impaired human epidermal growth factor receptor (HER3/ERBB3) and its catalytically active homologs EGFR and HER2 are essential for their signaling. Different ligands can activate these receptor pairs but lead to divergent signaling outcomes through mechanisms that remain largely unknown. We used stochastic optical reconstruction microscopy (STORM) with pair-correlation analysis to show that EGF and neuregulin (NRG) can induce different extents of HER3 clustering that are dependent on the nature of the coexpressed HER receptor. We found that the presence of these clusters correlated with distinct patterns and mechanisms of receptor phosphorylation. NRG induction of HER3 phosphorylation depended on the formation of the asymmetric kinase dimer with EGFR in the absence of detectable higher-order oligomers. Upon EGF stimulation, HER3 paralleled previously observed EGFR behavior and formed large clusters within which HER3 was phosphorylated via a noncanonical mechanism. HER3 phosphorylation by HER2 in the presence of NRG proceeded through still another mechanism and involved the formation of clusters within which receptor phosphorylation depended on asymmetric kinase dimerization. Our results demonstrate that the higher-order organization of HER receptors is an essential feature of their ligand-induced behavior and plays an essential role in lateral cross-activation of the receptors. We also show that HER receptor ligands exert unique effects on signaling by modulating this behavior.HER/ERBB receptors | receptor tyrosine kinase signaling | receptor clustering | STORM | EGFR activation T he human epidermal growth factor receptors (HERs/ErbBs) are essential regulators of development and adult homeostasis (1). All four of them, EGF receptor (EGFR; HER1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4), are at the focus of therapeutic efforts in a variety of human diseases. Most of what we know about their activation mechanism has been revealed by the studies on EGFR, which showed that ligand binding induces EGFR dimerization through a series of structurally well-defined interactions between the extracellular and intracellular receptor domains (2). These interactions result in the formation of an asymmetric kinase dimer in which one kinase (termed the "activator kinase") is asymmetrically positioned to activate the second kinase (termed the "receiver kinase") allosterically (3). The receiver kinase then is poised to phosphorylate the receptor tails, resulting in the recruitment of downstream signaling molecules and signal propagation.One of the characteristic features of the HER receptor family is a significant degree of heteromeric interactions in response to ligand binding through which the receptors activate a variety of signaling pathways (1). These interactions are particularly important for signaling by the orphan receptor HER2 and the catalytically impaired HER3, which do not signal on their own under normal conditions. Although all HER receptors are assumed to form h...

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“…In addition, as these approaches rely on ensemble measurements, they average over a large quantity so that information on the assembly state of particular protein subpopulations is lost. With the advent of single-molecule fluorescence microscopy techniques, it has become possible to systematically analyze the oligomeric state of individual membrane-bound proteins with minimal perturbation of the biological system (27). Notably, single-molecule photobleaching has emerged as a valuable tool to extract the stoichiometry of protein receptors, ion channels, and transporters at the plasma membrane of living cells (28).…”

mentioning

confidence: 99%

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

Cabukusta¹,

Köhlen²,

Richter

et al. 2016

Journal of Biological Chemistry

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Edited by George CarmanSingle-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Coimmunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.

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One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

Cited by 160 publications

References 54 publications

Model-independent counting of molecules in single-molecule localization microscopy

Model-independent counting of molecules in single-molecule localization microscopy

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

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