[Fluorescence photobleaching recovery; measurement of lateral movement of cell membrane components].
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Cell Membrane↗
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Measurement of the translational mobility of concanavalin A in glycerol-saline solutions and on the cell surface by fluorescence recovery after photobleaching.
The fluorescence recovery kinetics of succinyl-fluorescein Concanavalin A (S-F-ConA) in glycerol-physiological saline solutions of high viscosity and when bound to the surface of mouse fibroblasts were measured following brief photobleaching using a laser excited fluorescence microscope. In the high viscosity solutions, the recovery kinetics, interpreted on the basis of a simple diffusion model, yielded a diffusion coefficient in close agreement with the values predicted by the Stokes-Einstein equation. Recovery kinetics for S-F-ConA bound to the surface of mouse 3T3 and SV3T3 cells cultured in vitro yielded diffusion coefficients in the range of 5-10-10(-11) cm2/s, values considerably lower than those reported previously for membrane proteins. These measurements indicated that a considerable fraction of the S-F-ConA molecules bound to the cell surface are immobilized. These results are discussed in relation to current concepts of lateral motion of protein components within natural membranes.
Binding Sites↗
Membrane dynamics of differentiating cultured embryonic chick skeletal muscle cells by fluorescence microscopy techniques.
Changes in membrane fluidity during myogenesis have been studied by fluorescence microscopy of individual cells growing in monolayer cultures of embryonic chick skeletal muscle cells. Membrane fluidity was determined by the techniques of fluorescence photobleaching recovery (FPR), with the use of a lipid-soluble carbocyanine dye, and by fluorescence depolarization (FD), with perylene used as the lipid probe. The fluidity of myoblast plasma membranes, as determined from FPR measurements in membrane areas above nuclei, increased during the period of myoblast fusion and then returned to its initial level. The membrane fluidity of fibroblasts, also found in these primary cultures, remained constant. The fluidity in specific regions along the length of the myoblast membrane was studied by FD, and it was observed that the extended arms of the myoblast have the highest fluidity on the cell and that the tips at the ends of the arms had the lowest fluidity. However, since the perylene probe used in the FD experiments appeared to label cytoplasmic components, changes in fluidity measured with this probe reflect changes in membrane fluidity as well as in cytoplasmic fluidity. The relative change in each of these compartments cannot yet be ascertained. Tips have specialized surface structures, filopodia and lamellipodia, which may be accompanied by a more immobile membrane as well as a more rigid cytoplasm. Rounded cells, which may also have a more convoluted surface structure, show a lower apparent membrane fluidity than extended cells.
Animals↗
Mobility and distribution of a cell surface glycoprotein and its interaction with other membrane components.
Fluorescence photobleaching recovery and immunofluorescence methods have been used to study the lateral mobility and topographical distribution of a major cell surface glycoprotein (CSP). Both endogenous CSP and fluorescent-labeled exogenous CSP bind to the cell surface in a fibrillar pattern and are immobile on the experimental time scale. Azide, vinblastine, and cytochalasin B do not alter the immobility and cell surface distribution of the CSP molecules. Therefore, oxidative phosphorylation and the cytoskeleton do not seem to be responsible for the properties of the bound glycoprotein. The presence of immobile CSP fibrils does not, however, impede the diffusion of a lipid probe, a ganglioside analogue, or various surface antigens. Therefore, the fibrils apparently do not form a "barrier" across the lipid phase of the plasma membrane. In contrast, concanavalin A binds to CSP and is largely immobile in regions rich in CSP. The presence of immobile concanavalin A receptors in areas or on cells lacking CSP indicates that other types of immobile concanavalin A receptors also exist.CSP does not bind to lipid bilayers composed of phosphatidylcholine or oxidized cholesterol. It does bind to dextran-coated bilayers as a diffuse distribution of mobile molecules that can patch after addition of antibodies to CSP. The latter result suggests that CSP molecules do not interact strongly with other CSP molecules under these conditions. Exogenous CSP binds to regions on the cell surface that already bear CSP. In view of the apparent weakness of CSP-CSP interactions on the lipid bilayer, it seems possible that the assembly of CSP fibrils is nucleated by cell surface components in addition to CSP.
Antigens↗
Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin.
Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.
Adipose Tissue↗
Mobility and density of AgB, "Ia", and Fc receptors on the surface of lymphocytes from young and old rats.
Analysis of spleen cell populations from old Lewis rats (greater than 24 months) and from young Lewis rats (3 to 4 months) in a fluorescence-activated cell sorter indicated that with aging there is a loss of brightly stained Ia and Fc receptor- (FcR) positive cells. The density of AgB, Ia, and FcR was diminished on the surface of cells from old rats. The rate of capping of all three membrane proteins was slower on cells from old rats. Colchicine treatment allows capping of AgB with a single ligand only in young rats. Fluorescence photobleach recovery experiments (FPR) show that the fluidity of the lymphocyte membrane from old rats is diminished and the lateral diffusion of AgB is decreased. The colchicine and FPR experiments suggest that the changes in capping in old rats are due to, in part, alterations in membrane fluidity and cytoskeletal function.
Aging↗
Lipid domains in biological membranes: their structural and functional perturbation by free fatty acids and the regulation of receptor mobility. Co-presidential address.
We have studied the interaction of free fatty acids (FFAs) with cell membranes and lipid bilayers by monitoring changes in the emission polarization of the fluorescent probes diphenylhexatriene (DPH) and anilino-naphthalene sulfonate (ANS). We found that the FFAs readily intercalate into membranes and produce significant changes in the packing of the lipid molecules. The membrane alterations could be divided into two patterns: the cis-unsaturated FFAs (designated Group A) disorder the membranes' interior (as reported by DPH) and order the head group region (as reported by ANS); the trans-unsaturated or saturated FFAs (Group B) do not alter the bilayer interior but also order the head group region. Using solution theory, the shift in transition midpoint temperatures as a function of fatty acid type was used to infer that the Group A FFAs partition into fluid domains, while Group B FFAs partition preferentially into gel-like domains. These results are explained in terms of a domain model of membrane lipid structure. Low concentrations of Group A FFAs inhibit the capping of surface immunoglobulin (Ig), whereas no effect was seen with Group B FFAs. The capping inhibition caused by Group A FFAs was reversible with increasing doses of extracellular calcium. Fluorescence photobleaching recovery showed that the Group A FFAs do not inhibit receptor immobilization associated with patch formation but rather inhibit the final energy-dependent movement of the patched receptors into a cap. We have also shown that the Group A FFAs cause a shift in membrane-bound calcium to the lipid phase from probably protein calcium-binding sites. The data have generated a model of receptor mobility invoking a trans-membrane, calcium-binding, receptor-anchoring protein, linked to the cytoskeleton. Inhibition of capping by Group A FFAs is postulated to be due to perturbation of specific lipid domains associated with this protein, such perturbation leading to conformational changes in the protein, and consequent intramembraneous calcium sequestration in the lipid phase, rendering the calcium unavailable for activation of the cytoskeleton.
Calcium-Binding Proteins↗
Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes.
Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3'-dioctadecylindocarbocyanine) had a mean (+/-SD) diffusion coefficient of (1.7 +/- 0.3) x 10(-8) cm(2)/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of approximately 3 x 10(-10) cm(2)/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.
Animals↗
Receptor diffusion on cell surfaces modulated by locally bound concanavalin A.
In order to test the anchorage modulation hypothesis, the fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor mobility induced in 3T3 mouse fibroblasts by local binding of platelets labeled with concanavalin A (Con A). By measuring the diffusion of antibody-labeled cell surface receptors at various points on the cell surface, two states, immobile and mobile, were distinguished in the receptor population. Bound Con A-platelets, occupying between 4% and 30% of the cell surface, decreased the diffusion coefficient of the mobile population by a factor of 6. The magnitude of this effect was independent of distance from the sites of the bound Con A-platelets, demonstrating the propagated and nonlocal properties of the modulation effect. The immobile fraction of the population was not changed by Con A-platelet binding. Modulation of the diffusion constant of mobile receptors was partially reversed by treatment with microtubule-disrupting agents such as Colcemid and Vinca alkaloids. High doses of soluble Con A induced even higher levels of modulation than Con A-platelets, but reversal by microtubule-disrupting drugs was observed. These experiments provide additional support for the anchorage modulation hypothesis and provide a measure of the nature and degree of mobility at the molecular level. They also put important constraints on the hypothesized interactions among submembranous components (microtubules and microfilaments) of surface modulating assemblies.
Antigen-Antibody Complex↗
Fluorescence Loss After Photoactivation (FLAPh): A Pulse-Chase Cellular Assay for Understanding Kinetics and Dynamics of Viral Inclusions.
Influenza A virus (IAV) relies on host cellular machinery for replication. Upon infection, the eight genomic segments, independently packed as viral ribonucleoproteins (vRNPs), are released into the cytosol before nuclear import for viral replication. After nucleocytoplasmic transport, the resulting progeny vRNPs reach the cytosol, accumulating in highly mobile and dynamic viral inclusions that display liquid properties. Being sites postulated to support IAV genome assembly, the biophysical properties of IAV inclusions may be critical for function. In agreement, imposing liquid-to-solid transitions was demonstrated to impact viral replication negatively. Therefore, screening for host factors or compounds able to alter the material properties may provide the molecular basis for how influenza genomic complex forms as well as identify novel antivirals. Conventional techniques employed to investigate biomolecular condensates' material properties include fluorescence correlation spectroscopy, raster image correlation spectroscopy, single molecule or microrheology particle tracking, and Fluorescence Recovery After Photobleaching (FRAP). These approaches allow measuring molecular dynamics in systems that do not move very much. However, the analysis of highly mobile intracellular condensates, such as IAV inclusions, poses significant challenges as these structures not only constantly move within the cell but also exchange material, fusing, and dividing, rendering the quantitation of internal rearrangements and diffusion coefficients of molecules within condensates inaccurate. As an alternative, we opted for measuring the kinetics and the exchange of material between IAV inclusions using the Fluorescence Loss After Photoactivation (FLAPh) technique. It involves pulse photoactivation of individual or pools of viral inclusions in the cell, and chasing over time in photoactivated and non-photoactivated regions. This approach is suitable for quantifying the movement and spatial distribution of components within inclusions over time, enabling the determination of both the distance and speed from a specific cellular location. As a result, this method allows the quantification of decay profiles, half-lives, decay constant rate, and mobile and immobile fractions in viral inclusions. It, therefore, enables high throughput screenings for compounds or host factors that affect this dynamism and indirectly allows assessing the material properties of IAV inclusions.
Humans↗
Lateral mobility of an amphipathic apolipoprotein, ApoC-III, bound to phosphatidylcholine bilayers with and without cholesterol.
The technique of fluorescence recovery after photobleaching was used to investigate the lateral mobility of a fluorescein-labeled amphipathic apolipoprotein, ApoC-III, bound to multibilayers prepared from dipalmitoyl phosphatidylcholine, egg phosphatidylcholine, and a 1:1 (molar ratio) mixture of egg phosphatidylcholine and cholesterol. In dipalmitoyl phosphatidylcholine bilayers the lateral diffusion coefficient (D) for the protein is about 2 x 10(-9) cm(2) sec(-1) at 20 degrees C and about 9 x 10(-8) cm(2) sec(-1) at 45 degrees C. Plots of D versus temperature in this system show a transition between about 30 and 35 degrees C. Arrhenius activation energies for the diffusion in this case between 15 and 30 degrees C and between 35 and 45 degrees C are 28.5 and 7.0 kcal mol(-1), respectively (1 calorie = 4.18 joules). In egg phosphatidylcholine bilayers, D is about 3 x 10(-8) cm(2) sec(-1) at 20 degrees C and the Arrhenius activation energy for diffusion is 8.1 kcal mol(-1) between 15 and 35 degrees C in this system. In bilayers prepared from an equimolar mixture of egg phosphatidylcholine and cholesterol D at 20 degrees C is about 1.4 x 10(-9) cm(2) sec(-1) and the Arrhenius activation energy for the diffusion of the protein in this system between 15 and 35 degrees C is 15.1 kcal mol(-1). Light-scattering and fluorescence-polarization results indicate that binding of this protein does not affect the gel-to-liquid crystalline phase transition of bilayer membranes but does mediate a major, reversible aggregation of the vesicles at about 33 degrees C. These results lend support to the view that ApoC-III resides in the head-group region of the bilayer and suggest that its lateral diffusion coefficient represents an upper bound for integral membrane proteins.
Apolipoproteins↗
Bridging-driven condensation by eukaryotic SMC complexes is a conserved feature of genome organization.
The Structural Maintenance of Chromosome (SMC) protein family plays a central role in higher-order genome organization through ATP-dependent DNA loop extrusion by cohesin and condensin and other processes. Whether these activities fully account for the complexity of chromosome architecture remains unknown. Here, we uncover a conserved ATP-independent mechanism of chromatin condensation by SMC complexes, occurring via biomolecular condensation. Using single-molecule fluorescence imaging, we show that a variety of SMCs form dynamic DNA-bound condensates that exhibit key features of biomolecular condensates, including droplet coalescence, fluorescence recovery after photobleaching, and rapid exchange with free SMC complexes. Atomic force microscopy analysis of human cohesin-DNA assemblies reveals DNA-length-dependent clustering, providing evidence for bridging-driven condensation. Analyses of in vivo super-resolution imaging and high-throughput chromosome conformation capture (Hi-C) data indicate that these condensates form chromatin-associated clusters with multi-loop structures. Together, our results establish that SMC complexes employ ATP-independent phase condensation as well as ATP-dependent activities to shape genome architecture. This work reveals a broadly conserved principle of chromosomal organization across eukaryotes.
Chromosomal Proteins, Non-Histone↗
A fluorescence approach of the determination of translational diffusion coefficients of lipids in phospholipid monolayer at the air-water interface.
In the present work, it is shown that the photobleaching technique as well as experimentation based on fluorescence recovery after bleaching can be extended to monolayers spread at the air-water interface. A mathematical model is derived which allows the determination of translational diffusion coefficients of species diffusing in such a system. Using 12-(9-anthroyl)stearic acid (anthroylstearate) as a fluorescent probe, dispersed either in dipalmitoylphosphatidylcholine or in dipalmitoylphosphatidylglycerol in various conditions of subphase ionic composition and surface pressure of the monolayer, including phase transition domains, we are led to the following conclusions: 1. Anthroylstearate molecules seem to aggregate in 'microdomains' where their fluorescence properties remain unchanged regardless of the compression states of the host monolayer. 2. In any case, a break in the diffusion constants appears on compressing films of both dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. In particular, this break coincides with the liquid expanded to gel phase transition of these lipids when it occurs. 3. Diffusion of anthroylstearate in dipalmitoylphosphatidylglycerol depends strongly on the subphase ionic strength and on the nature of cations: Na+, Mg2+, Ca2+.
Diffusion↗
Photobleaching recovery studies of membrane events accompanying lectin stimulation of rabbit lymphocytes.
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Animals↗