Fluorescence Energy Transfer Techniques Research Articles

Ultra-photostable DNA fluorocubes: mechanism of photostability and compatibility with FRET and dark quenching

DNA-based FluoroCubes were recently developed as a solution to photobleaching, a ubiquitous limitation of fluorescence microscopy (Niekamp, Stuurman, & Vale, Nature Methods, 2020). A FluoroCube is a compact ∼4×4×5.6 nm3 four-helix bundle of synthetic DNA that has six fluorophores attached to it. FluoroCubes exhibit remarkable photostability, remaining fluorescent up to ∼50× longer than single fluorophores under constant illumination and emitting up to ∼40× as many photons in total. This work seeks to answer two important questions about FluoroCubes: First, what is the mechanism by which photostability is enhanced? Second, how compatible are FluoroCubes with fluorescence energy transfer techniques? The answer to the first question is crucial to our understanding of FluoroCube design and the chemical physics of photobleaching, while the second question relates to the application space of FluoroCubes.

The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex.

CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique. Our results show that Cas9–sgRNA complex divides the target DNA into several distinct domains: protospacer adjacent motif, linker, Seed, Middle and Tail. After seed pairing, the Cas9 transiently retains a semi-active conformation and induces the cleavage of either target or non-target strand. smFRET studies demonstrate that an intermediate state exists in prior to the formation of the fully stable R-loop complex. Kinetics analysis of this new intermediate state indicates that the lifetime of this state increases when the base-pairing length of guide-DNA hybrid duplex increases and reaches the maximum at the size of 18 bp. These data provide new insights into the process of R-loop formation and reveal the source of off-targeting in CRISPR/Cas9 system.

Abstract 626: Interaction Between The Ang-(1-7) Receptor Mas And The Bradykinin B2 Receptor: Functional Implications

G protein-coupled receptors (R) exist as homo- or hetero-oligomers, which is essential for receptor function. Since BK actions were blocked by a Mas R antagonist or that Ang-(1-7) responses disappeared when the BK receptor B2 was blocked, we hypothesized that Mas and B2 Rs on the plasma membrane may interact through hetero-oligomer formation. Our aim was to investigate the existence of heteromerization between Mas and B2 Rs by the fluorescence energy transfer (FRET) technique and the functional consequences of this oligomer formation. HEK293T cells were transfected with the coding sequence for Mas R fused to YFP and B2 R fused to CFP. After 48 h cells were incubated in the absence and presence of 1 μM Ang-(1-7) or BK during 15 min and interaction between Mas and B2 R was evaluated by FRET. Functional consequences of this interaction were determined by ligand binding assays. A positive FRET was observed in cells cotransfected with MasR-YFP and B2R-CFP, suggesting that both Mas and B2 Rs interact by a hetero-oligomer formation in a constitutive manner. This hetero-oligomer was not altered by the agonist because FRET was not modified when the cells were stimulated with BK or Ang-(1-7). Ang-(1-7) or BK induced internalization of this hetero-oligomer into early endosomes since MasR-YFP or B2R-CFP colocalized with Rab-5, an early endosome marker, after ligand stimulation. When MasR-YFP plus B2R-CFP transfected cells were stimulated with Ang-(1-7) there was a decrease of 82±6% in Mas R and 58±4% in B2 R present in the plasma membrane. Conversely, when MasR-YFP plus B2R-CFP transfected cells were stimulated with BK there was a decrease of 91±4% in B2 R and 53±3% in Mas R in the plasma membrane. This result clearly demonstrates that in co-expressing cells of both receptors the selective stimulation of one of the GPCRs promotes co-internalization of both receptors. We conclude that Mas and B2 Rs constitutively interact through an hetero-oligomer formation at the plasma membrane which may explain the cross-talk between Ang-(1-7) and BK. This hetero-oligomer is internalized upon stimulation with either Ang-(1-7) or BK, leading to a decrease in the number of Rs present in the membrane.

Single Molecule Observation of Cyclization of Short DNA Duplex

In the presented work, a single molecule DNA cyclization assay was used to follow the looping kinetics of single DNA 83 bp molecules, utilizing single molecule fluorescence energy transfer (smFRET) technique. The assay was first prepared in a Na+ free condition and the majority of the DNA was in its unlooped form. A sudden Na+ jump was introduced at different concentrations (0.05-1.75M) and finally yielded DNA in its looping state by annealing the complementary single-strand overhangs of the assay. Looping and unlooping rates were obtained from the kinetic measurements. The result shows a positive and negative linear dependence of the Na+ concentration to the looping and unlooping rate, respectively, until they reach a plateau at 500 mM. The plateau persists until about 1M. For concentrations beyond 1M, an immoderate increase in looping rate is noticed while the unlooping rate does so gradually. Above 1M Na+ there is a preference of looping events that is attributed to the increase of the annealing rate of the overhangs rather than increased flexibility, consistent with earlier studies by Ibrahim Cisse et al. (2012).A protein mediated cyclization assay was also used in experiments with HU protein in which a dramatic increase in the looping rate is noticeable. However in high HU concentration, looping is prohibited implying filament formation.

α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant, AHSPV56G

A kinetic analysis has been made of the interaction of alpha-Hb chains with a mutant alpha-hemoglobin stabilizing protein, AHSP(V56G), which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent alpha chains until final binding to the partner beta-Hb. Rather than protecting alpha chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with alpha-Hb. For both AHSP(WT) and AHSP(V56G), the binding to alpha-Hb is quite rapid relative to the alpha-beta reaction, as expected because the chaperone binding must be quite competitive to complete the alpha chain folding process before alpha-Hb binds irreversibly to beta-Hb. The main kinetic difference is a dissociation rate of AHSP(V56G).alpha-Hb some four times faster relative to AHSP.alpha-Hb. Considering a role of protein folding, the AHSP(V56G) apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP.alpha-Hb + beta-Hb --> AHSP + alphabeta) can be quite long, especially if there is an excess of AHSP relative to beta-Hb monomers.

Essential Role for the CRAC Activation Domain in Store-dependent Oligomerization of STIM1

Oligomerization of the ER Ca(2+) sensor STIM1 is an essential step in store-operated Ca(2+) entry. The lumenal EF-hand and SAM domains of STIM1 are believed to initiate oligomerization after Ca(2+) store depletion, but the contributions of STIM1 cytosolic domains (coiled-coil 1, CC1; coiled-coil 2, CC2; CRAC activation domain, CAD) to this process are not well understood. By applying coimmunoprecipitation and fluorescence photobleaching and energy transfer techniques to truncated and mutant STIM1 proteins, we find that STIM1 cytosolic domains play distinct roles in forming both "resting" oligomers in cells with replete Ca(2+) stores and higher-order oligomers in store-depleted cells. CC1 supports the formation of resting STIM1 oligomers and appears to interact with cytosolic components to slow STIM1 diffusion. On store depletion, STIM1 lacking all cytosolic domains (STIM1-DeltaC) oligomerizes through EF-SAM interactions alone, but these oligomers are unstable. Addition of CC1 + CAD, but not CC1 alone, enables the formation of stable store-dependent oligomers. Within the CAD, both CC2 and C-terminal residues contribute to oligomer formation. Our results reveal a new function for the CAD: in addition to binding and activating Orai1, it is directly involved in STIM1 oligomerization, the initial event triggering store-operated Ca(2+) entry.

Dimerization in the absence of higher-order oligomerization of the G protein-coupled secretin receptor

Oligomerization of G protein-coupled receptors has been proposed to affect receptor function and regulation; however, little is known about the molecular nature of such complexes. We previously utilized bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic Family B secretin receptor can form oligomers. We now explore the order of oligomerization present utilizing unique bimolecular fluorescence complementation and energy transfer techniques. The non-fluorescent carboxyl-terminal and amino-terminal halves of yellow fluorescent protein (YFP) were fused to the carboxyl terminus of the secretin receptor. These constructs bound secretin normally and signaled in response to secretin like wild type receptor. When co-expressed on COS cells, these constructs physically interacted to yield typical YFP fluorescence in biosynthetic compartments and at the plasma membrane, reflecting receptor homo-dimerization. However, the addition of another potential partner in form of Rlu- or CFP-tagged secretin receptor yielded no significant BRET or FRET signal, respectively, under conditions in which intact YFP-tagged secretin receptor yielded such a signal. Absence of higher-order receptor oligomers was further confirmed using saturation BRET techniques. Absence of significant resonance transfer to the secretin receptor homo-dimer was true for carboxyl-terminally-tagged secretin receptor, as well as for receptor incorporating the transfer partner into each of the three distinct intracellular loop domains. These results suggest that the secretin receptor can exist only as a structurally-specific homo-dimer, without being present as higher-order oligomers.

Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex

Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n = 10(±2) base-pairs, which is significantly shorter than the total site-size of 16(±2) nucleotides of the enzyme–ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNA-binding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5′ or 3′ end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the non-catalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein–protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed.

Investigation of the first shot phenomenon in MALDI mass spectrometry of protein complexes

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of noncovalent protein complexes is difficult, due to the disruptive nature of processes occurring during MALDI sample preparation and ion formation. Sometimes the observation of intact noncovalent protein complexes with MALDI is only possible if data are acquired from the first laser shot fired at a fresh sample; this is called the 'first shot phenomenon'. To study the origin of the first shot phenomenon, we used MALDI-MS and confocal laser scanning microscopy (CLSM) to examine typical MALDI sample preparations with embedded protein complexes, labeled with fluorophores. Fluorescence energy transfer techniques allowed the differentiation between intact and dissociated protein complexes with CLSM. In cases where a first shot behavior was observed by MALDI-MS, it was found to be accompanied by localization of protein complexes at the exterior of the sample crystals. Segregation of the large protein complexes to the exterior and dissociation of the complexes in the crystal interior during sample crystallization can rationalize this observation.

Spectroscopic and catalytic studies of lipases in ternary hexane–1-propanol–water surfactantless microemulsion systems

A series of water-in-oil microemulsion systems formulated without surfactant were used to solubilize lipases from Rhizomucor miehei and Candida antarctica B. The effect of the system's composition on the velocity of enzymic reactions was investigated following a model esterification reaction. The interaction between enzymes and the microemulsion environment was studied by steady state fluorescence spectroscopy. The site of localization of the enzyme within the different microdomains of the dispersed phase was investigated by applying the fluorescence energy transfer technique. To determine the properties of the interface between water and organic solvent of the surfactantless microemulsion systems the Electron Paramagnetic Resonance (EPR) spectroscopic technique was applied. The results indicated that even at low water content, water-rich structures are formed. This was confirmed by conductivity measurements. By the addition of enzyme it was observed that when the aqueous phase of the surfactantless microemulsion systems exceeds 2% (v/v) the enzyme retains its catalytic activity, as it is located within the water pools that protect it from the organic solvent. These confined water phases show a propanol rich interface with hexane and their structure depends on the system's composition.

Studies on Protein–[60]Fullerene Interactions: The Lysozyme–[60]Fullerene Model System

A lysozyme–[60]fullerene adduct was synthesized and isolated for the first time. This adduct was water‐soluble and showed strong interaction between lysozyme and [60]fullerene and was characterized by UV‐VIS and fluorescence energy transfer technique. Keeping possible biomedical applications of [60]fullerene in view, the protein–[60]fullerene interactions were studied taking lysozyme as a model protein.

Interactions of the Escherichia coli DnaB Helicase Hexamer with the Replication Factor the DnaC Protein. Effect of Nucleotide Cofactors and the ssDNA on Protein–Protein Interactions and the Topology of the Complex

Quantitative studies of interactions between the Escherichia coli replication factor DnaC protein and the DnaB helicase have been performed using sedimentation velocity and fluorescence energy transfer techniques. The applied novel analysis of the sedimentation data allows us to construct thermodynamic rigorous binding isotherms without any assumption as to the relationship between the observed molecular property of the complexes formed, the average sedimentation coefficient, or the degree of binding. Experiments have been performed with the fluorescein-modified DnaB helicase, which allows an exclusive monitoring of the DnaB–DnaC complex formation. The DnaC binding to the unmodified helicase has been characterized in competition experiments. The data establish that, in the presence of the ATP analog AMP-PNP, or ADP, a maximum of six DnaC monomers bind cooperatively to the DnaB hexamer. The positive cooperative interactions are limited to the two neighboring DnaC molecules. Analyses using a statistical thermodynamic hexagon model indicate that, under the solution conditions examined, the affinity is characterized by the intrinsic binding constant K=1.4(±0.5)×10 5 M −1 and cooperativity parameter σ=21±5. These data suggest strongly that the DnaC–DnaB complex exists in vivo as a mixture of complexes with a different number of bound DnaC molecules, although the complex with six DnaC molecules bound dominates the distribution. The DnaC nucleotide-binding site is not involved in the stabilization of the complex. Moreover, the hydrolysis of NTP bound to the helicase or the DnaC is not required for the release of the DnaC protein from the complex. The single-stranded DNA (ssDNA) bound to the helicase does not affect the DnaC protein binding. However, in the presence of the DNA, there is a significant difference in the energetics and structure of the ternary complex, DnaC–DnaB–ssDNA, formed in the presence of AMP-PNP as compared to ADP. The topology of the ternary complex DnaC–DnaB–ssDNA has been determined using the fluorescence energy transfer method. In solution, the DnaC protein-binding site is located on the large 33 kDa domain of the DnaB helicase. The significance of the results in the functioning of the DnaB helicase–DnaC protein complex is discussed.

Rat polymerase beta binds double-stranded DNA using exclusively the 8-kDa domain. Stoichiometries, intrinsic affinities, and cooperativities.

Analyses of the interactions of rat polymerase beta (rat pol beta) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol beta binds to dsDNA oligomers with the site-size of the enzyme-dsDNA complex n = 5 +/- 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 A from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA. Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state. The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg(2+) cations to the protein. Surprisingly, binding of rat pol beta to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol beta complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein-protein interactions. The significance of these results for the pol beta functioning in damaged-DNA recognition processes is discussed.

Nanometre-scale structure of fluid lipid membranes

The lipid-bilayer component of biological membranes is a two-dimensional liquid under physiological conditions. Computer-simulation calculations based on statistical mechanical models predict this liquid to exhibit structure in the form of fluctuating lipid domains in the nanometre range. Indirect and direct experimental evidence for the small-scale structure is provided by fluorescence energy-transfer techniques and atomic-force microscopy, respectively.

Membrane lipid a-crystallin interaction and membrane Ca2+-ATPase activities

Purpose. To determine the effect of a-crystallin binding on lens membrane lipid characteristics and the stability of Ca2+-ATPase activity when challenged with H2O2 or elevated temperatures. Methods. a-Crystallin binding to muscle sarcoplasmic reticulum membranes was quantified using a centrifugation protocol. a-Crystallin binding to lens epithelial lipids was measured by a fluorescence energy transfer technique. Lipid phase transition temperature and lipid order was measured using fluorescence spectroscopy. Ca2+-ATPase activity was measured using classical biochemical assays. Results. The main phase transition temperatures of multilamellar vesicles composed of sphingomyelin or lipids extracted from bovine lens were 40°C and 20°C, respectively. In the presence of saturating amounts of a-crystallin, the phase transition temperature and lipid order of both sphingomyelin and lens lipid membranes remained almost the same as that without a-crystallin. The interaction of a-crystallin and lipid is likely to be restricted to the membrane surface. The binding of a-crystallin did not influence the oxidative or thermal inactivation of the Ca2+-ATPase pump. Conclusion. a-Crystallin-lens membrane binding does not protect the Ca2+-ATPase pump from thermal derangement or oxidation by H2O2.

Functional and Structural Heterogeneity of the DNA Binding Site of the Escherichia coli Primary Replicative Helicase DnaB Protein

The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude approximately 10 nucleotides. ssDNA affinity for the strong subsite is approximately 3 orders of magnitude higher than that for the weak subsite. Fluorescence energy transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5'-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.

Binding mode of porphyrins to poly[d(A-T) 2] and poly[d(G-C) 2

We examined the binding geometry of Co- meso-tetrakis ( N-methyl pyridinium-4-yl)porphyrin, Co- meso-tetrakis ( N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T) 2] and poly[d(G-C) 2] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C) 2] and positive when bound to poly[d(A-T) 2]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T) 2], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C) 2], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T) 2] and poly[d(G-C) 2], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.

Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers

A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and in terms of nanometer-scale lipid-domain formation.

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.

Distinct association of transferrin receptor with HLA class I molecules on HUT-102B and JY cells

The topological relationship of transferrin receptor (TfR) has been studied relative to the heavy and light chains of the HLA class I molecules, class II molecules, interleukin-2 receptor α-chain and ICAM-1 molecule in the plasma membrane of HUT-102B2 T and JY B lymphoblastoid cell lines using the flow cytometric fluorescence energy transfer technique (FCET). The effect of different growing conditions (logarithmic and plateau phases) on the relative surface density of the receptors and the lateral organization of the TfR was also studied. The TfR showed a high degree of self-association on the surface of both cell lines regardless of the growing phase. TfR was in close vicinity to HLA class I heavy and light chains on HUT-102B cells in both plateau and logarithmic phases, while it was not associated with HLA class I on the surface of JY cells. HLA class II molecules form a cluster with TfR on HUT-102B cells, while only a modest association was found on JY cells, and only in the logarithmic phase. The possible explanation of this distinct association and a two dimensional model of the antigen and receptor distributions are presented in this paper.