Homo‐FRET Imaging as a Tool to Quantify Protein and Lipid Clustering

Bader, Arjen N.; Hoetzl, Sandra; Hofman, Erik; Voortman, Jarno; Henegouwen, Paul M.P. van Bergen en; Meer, Gerrit van; Gerritsen, Hans C.

doi:10.1002/cphc.201000801

Cited by 86 publications

(82 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Homotransfer FRET assesses energy transfer between identical fluorescent donor and acceptor molecules by measuring fluorescence anisotropy as probe molecules are photobleached (20). Increasing anisotropy as probe molecules are progressively photobleached is indicative of FRET.…”

Section: Methodsmentioning

confidence: 99%

Restricted Lateral Diffusion of Luteinizing Hormone Receptors in Membrane Microdomains

Wolf‐Ringwall

Winter

Liu

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Signal transduction by luteinizing hormone (LH)3 receptor plays an essential role in the normal reproductive function of both male and female mammals by promoting ovulation, follicle maturation, corpus luteum formation, and steroidogenesis. LH receptor signaling in response to saturating concentrations of LH or human chorionic gonadotropin (hCG) involves substantial changes in receptor lateral and rotational dynamics (1, 2) as well as the association of LH receptors with membrane rafts (3). FRET techniques (4) and electron microscopy (5) indicate that functional hormone-receptor complexes can also become self-associated into dimers/oligomers following ligand binding. Previous experimental strategies have examined changes in receptor motions on collections of cells or large numbers of fluorescently tagged molecules on single cells. It is now possible, however, to examine the lateral motions of individual LH receptors on living cells using microscope-based single particle tracking techniques (6).Although the mechanism involved in retention of hCG-occupied LH receptors in small membrane compartments is still unclear, exposure to saturating concentrations of hCG results in confinement of the majority of LH receptors within small cell surface compartments. LH receptors remain within these compartments for comparatively long times and appear to diffuse pseudo-randomly before being captured within another compartment of similar size (7). Similar behavior has been described and analyzed by Kusumi and co-workers (6) for selected phospholipids and for transferrin receptors (8) and by Daumas and co-workers (9) for the -opioid receptor. Daumas argues that the -opioid receptor can both diffuse within the bulk membrane and exhibit confinement within a microdomain that itself diffuses slowly. Kusumi and co-workers (6) suggest that particles may be confined by proteins forming a barrier that is either continuous or discontinuous, leading to receptor diffusion within small membrane regions accompanied by intermittent escape from compartments and periods of unrestricted diffusion in the bulk membrane.Our previous studies of LH receptor lateral and rotational diffusion suggest that actin microfilaments or membrane protein interactions with the cytoskeleton may provide organizing structures that restrict the lateral motions of receptors (1, 2). However, the mechanism of hormone-initiated LH receptor confinement in small compartments has not been examined previously in detail at the single molecule level. In the present work, we compared the lateral dynamics of individual LH receptors that were either FLAG-tagged and identified using FLAG-specific antibodies with the diffusion of native receptors occupied by gold nanoparticle-hCG conjugates (Au-hCG) or gold-deglycosylated hCG (Au-DG-hCG). The latter material is

show abstract

Section: Methodsmentioning

confidence: 99%

Restricted Lateral Diffusion of Luteinizing Hormone Receptors in Membrane Microdomains

Wolf‐Ringwall

Winter

Liu

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…To investigate whether the anti-EGFR VHHs induce receptor clustering, we have used homo-Förster resonance energy transfer (homo-FRET) (Bader et al, 2011). In this assay, clustering of EGFR is analyzed on NIH 3T3 cells expressing EGFR fused to monomeric GFP (mGFP) by confocal time-resolved fluorescence anisotropy imaging microscopy.…”

Section: Biparatopic Vhhs Induce Egfr Clusteringmentioning

confidence: 99%

EGFR endocytosis requires its kinase activity and N-terminal transmembrane dimerization motif

Heukers

Vermeulen

Fereidouni

et al. 2013

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

Summary EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.

show abstract

“…71,73,83 In-cell studies of the K-Ras lipid anchor using Homo-FRET and electron microscopy also demonstrate that the lipid anchor is sufficient for dimerization and clustering on the membrane. 84 How can the HVR, -that is not involved in nucleotide binding or hydrolysis -affect dimer and nanocluster formation in a nucleotide dependent manner? It is possible that the catalytic domain of Ras has a way to communicate with the HVR.…”

Section: Ras Dimerization Oligomerization and Clusteringmentioning

confidence: 99%