Resonance energy transfer studies of the mechanisms of microclustering of lentil lectin membrane receptors on HeLa cells

Schreiber, Alain A.B.; Hoebeke, Johan; Vray, Bernard; Strosberg, A. Donny

doi:10.1016/0014-4827(81)90103-8

Cited by 10 publications

(2 citation statements)

References 18 publications

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“…The same separation distance was found in the microaggregated state and the visible patches. Microaggregation proved to be reversible while no dissociation of the visible patches could be observed on the experimental time scale (Schreiber et al 1981). The FRET efficiency was found to be essentially zero with prefixed cells, indicating that the lectin binding was responsible for the microclustering.…”

Section: Biological Significance Of Fret and Fcet Measurementsmentioning

confidence: 86%

Luminescence spectroscopic approaches in studying cell surface dynamics

Matkó

Szöllősi

Trón

et al. 1988

Quart. Rev. Biophys.

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The major elements of membranes, such as proteins, lipids and polysaccharides, are in dynamic interaction with each other (Albertset al.1983). Protein diffusion in the lipid matrix of the membrane, the lipid diffusion and dynamic domain formation below and above their transition temperature from gel to fluid state, have many functional implications. This type of behaviour of membranes is often summarized in one frequently used word membrane fluidity (coined by Shinitzky & Henkart, 1979). The dynamic behaviour of the cell membrane includes rotational, translational and segmental movements of membrane elements (or their domain-like associations) in the plane of, and perpendicular to the membrane. The ever changing proximity relationships form a dynamic pattern of lipids, proteins and saccharide moieties and are usually described as ‘cell-surface dynamics’ (Damjanovichet al.1981). The knowledge about the above defined behaviour originates from experiments performed mostly on cytoplasmic membranes of eukaryotic cells. Nevertheless numerous data are available also on the mitochondrial and nuclear membranes, as well as endo (sarco-)plasmic reticulum (Martonosi, 1982; Slater, 1981; Siekevitz, 1981).

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Section: Biological Significance Of Fret and Fcet Measurementsmentioning

confidence: 86%

Luminescence spectroscopic approaches in studying cell surface dynamics

Matkó

Szöllősi

Trón

et al. 1988

Quart. Rev. Biophys.

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“…The quenching of the donor (F-4H7.1) fluorescence of doubly labeled vesicles and their detergent extract was measured using a spectrofluorometer (SFM 25; Kontron Analytical, Everett, MA) at 6°C as described by Schreiber et al (36). The excitation wavelength was 488 nm and the emission wavelength was 53(I nm.…”

Section: Fluorescence Energy Transfer Experimentsmentioning

confidence: 99%

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer.

Gorvel¹,

Mishal²,

Liegey³

et al. 1989

The Journal of Cell Biology

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Abstract. Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by crosslinking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.

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Flow cytometric measurements of fluorescence energy transfer using single laser excitation

et al. 1987

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Flow cytometric energy transfer (FCET)measurements between labeled specific sites of cell surface elements (Szollosi et al., Cytometry, 5210-216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: 1) The labeled ligands can be applied in excess, without washing, thereby enabling the investigation of relatively labile receptor-ligand complexes. 2) Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal.3) The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. 4) In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell-by-cell basis, offering data on intramolecular conformational changes.This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.Key terms: Resonance energy transfer, concanavalin A, Gross virus leukemia, flow cytometry, flow microfluorimetry, receptor densityThe cytoplasmic cell surface membrane plays a major role in communication between the environment and the cell interior. The cell senses specific external stimuli through surface receptors. The information necessary for cell function can be communicated by the aggregation state of these receptors or by changes in the distribution state of receptors on the membrane.The proximity relationship of membrane receptors can be studied by measuring the efficiency of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores conjugated to components in question (21). FRET measurements have been performed on cell surfaces using spectrofluorimetry (4,lB-201, microscopy (91, and flow cytometry (3,5,10,(13)(14)(15)24,25). The FRET efficiencies determined using spectrofluorimeters are values averaged over the entire homogeneous or heterogeneous population. The data obtained with spectrofluorimeters are highly perturbed by the amount of debris, dead cells, aggregates, etc. Furthermore the procedures used to eliminate excess labeled ligands also remove cells from the samples and present a n almost insurmountable problem when comparing fluorescence intensities of single-labeled samples with those of double-labeled samples. We have developed and recently published (5,24,25) a new method using a flow cytometer capable of dual-wavelength excitation to determine FRE...

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Resonance energy transfer studies of the mechanisms of microclustering of lentil lectin membrane receptors on HeLa cells

Cited by 10 publications

References 18 publications

Luminescence spectroscopic approaches in studying cell surface dynamics

Luminescence spectroscopic approaches in studying cell surface dynamics

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer.

Flow cytometric measurements of fluorescence energy transfer using single laser excitation

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