Fluorescent bio-barcode DNA assay for the detection of Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis is one of the most frequently reported causes of foodborne illness. It is a major threat to the food safety chain and public health. A highly amplified bio-barcode DNA assay for the rapid detection of the insertion element (Iel) gene of Salmonella Enteritidis is reported in this paper. The biosensor transducer is composed of two nanoparticles: gold nanoparticles (Au-NPs) and magnetic nanoparticles (MNPs). The Au-NPs are coated with the target-specific DNA probe which can recognize the target gene, and fluorescein-labeled barcode DNA in a 1:100 probe-to-barcode ratio. The MNPs are coated with the 2nd target-specific DNA probe. After mixing the nanoparticles with the 1st target DNA, the sandwich structure (MNPs-2nd DNA probe/Target DNA/1st DNA probe-Au-NPs-barcode DNA) is formed. A magnetic field is applied to separate the sandwich from the unreacted materials. Then the bio-barcode DNA is released from the Au-NPs. Because the Au-NPs have a large number of barcode DNA per DNA probe binding event, there is substantial amplification. The released barcode DNA is measured by fluorescence. Using this technique, the detection limit of this bio-barcode DNA assay is as low as 2.15 x 10(-16)mol (or 1 ng/mL).     

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.     

Intracellular delivery of nanometric DNA particles via the folate receptor

The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C(14)COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = -45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C(14)COrn)(2) lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE-PEG-folate. Physicochemical measurements showed particles coated with 2% of DPPE-PEG(3400)-folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy.     

Numerical Simulation of Broadband Scattering by Coated and Noncoated Metal Nanostructures Using Discrete Dipole Approximation Method

We suggest numerical method to study the optical response of metal nanostructures. The analysis of optical properties such as scattering and absorption by coated and noncoated nanogeometry has been done using discrete dipole approximation (DDA) method. The core-shell nanogeometry supports surface plasmon resonances, which are highly tunable from 400 to 1100 nm. The tunability of surface plasmon resonance (SPR) highly depends on the structural anisotropy and chosen core-shell material. Further, we have observed that aspect ratio is one of the key parameter to decide the nature and position of the plasmonic peaks and magnitude of optical cross section. We have also shown that coated nanospheroid is a more appropriate geometry as compared to coated nanosphere and noncoated nanospheroid in terms of wide tunability of surface plasmon resonance. The wide tunability in SPR is observed for the effective radii 90 nm core-shell (Au@SiO₂) nanospheroid with aspect ratio 0.1.     

Hydrocolloid coating of Xenopus laevis embryos

A novel technology for coating single cells and embryos with thin hydrocolloid (water-soluble polymer) films has been invented and patented. Coating is different from entrapment and immobilization in that the coating around the cell is thinner, comprising only a small fraction of the cell or embryo's diameter. Xenopus laevis embryos were coated with thin films of low-methoxy pectin (LMP), alginate, and iota- and kappa-carrageenans. These gums have different compositions and structures and as such created different coatings around the fertilized cells. All coated embryos appeared to develop normally, similar to noncoated embryos. Elemental detection by ICP-AES spectroscopy revealed that the embryo can control the diffusion of excess ions to which it is exposed during the coating process. The coatings delayed hatching by 18-24 h. Consequently, at hatch the embryos were at a more developed stage than their noncoated counterparts. The hydrocolloid coating reduced the thickness of the natural jelly coating (JC). With the iota-carrageenan coating, percent hatch was maximal, while with LMP it was minimal, as a result of the films' mechanical properties and thicknesses. LMP and alginate created smoother coatings than the carrageenans. Potential interactions between the coating and the natural JC are hypothesized. Overall, coatings appear to be a suitable tool for laboratories interested in performing longer-term experiments with embryos.     

Morphogenesis of bacteriophage Lambda: Electron microscopy of thin sections

Quantitative measurements on number, size, shape, location and time of appearance of heads and head-related structures in thin sections of induced bacteriophage λ lysogens were performed. Three types of particles can be distinguished: empty heads with a mean diameter of 39 nm (petit λ), heads partially filled with DNA with a mean diameter of 51 nm (grizzled particles) and particles filled with DNA, having a diameter of 47 nm (black particles). Some of the latter ones can be seen with a tail attached. The particles first to appear are the petit λ. A few minutes later grizzled and black particles can be seen. This sequence correspons to measurements of biological activities in lysates, i.e. to plaque-forming units, and to the number of particles which can be packaged with DNA and transformed in vitro to plaque-forming particles, respectively.DNA packaging seems to occur on the boundary area between cytoplasm and DNA plasm. Tails, on the other hand, accumulate near the cytoplasmic membrane.Two steps in DNA packaging can be distinguished, since one type of mutant blocked in DNA packaging (amber in gene A) produces paracrystalline agglomerations of petit λ and clusters of tails while another (amber in gene D) produces grizzled particles in addition.     

Syncytial-type cell plates: a novel kind of cell plate involved in endosperm cellularization of Arabidopsis

Cell wall formation in the syncytial endosperm of Arabidopsis was studied by using high-pressure-frozen/freeze-substituted developing seeds and immunocytochemical techniques. The endosperm cellularization process begins at the late globular embryo stage with the synchronous organization of small clusters of oppositely oriented microtubules ( approximately 10 microtubules in each set) into phragmoplast-like structures termed mini-phragmoplasts between both sister and nonsister nuclei. These mini-phragmoplasts produce a novel kind of cell plate, the syncytial-type cell plate, from Golgi-derived vesicles approximately 63 nm in diameter, which fuse by way of hourglass-shaped intermediates into wide ( approximately 45 nm in diameter) tubules. These wide tubules quickly become coated and surrounded by a ribosome-excluding matrix; as they grow, they branch and fuse with each other to form wide tubular networks. The mini-phragmoplasts formed between a given pair of nuclei produce aligned tubular networks that grow centrifugally until they merge into a coherent wide tubular network with the mini-phragmoplasts positioned along the network margins. The individual wide tubular networks expand laterally until they meet and eventually fuse with each other at the sites of the future cell corners. Transformation of the wide tubular networks into noncoated, thin ( approximately 27 nm in diameter) tubular networks begins at multiple sites and coincides with the appearance of clathrin-coated budding structures. After fusion with the syncytial cell wall, the thin tubular networks are converted into fenestrated sheets and cell walls. Immunolabeling experiments show that the cell plates and cell walls of the endosperm differ from those of the embryo and maternal tissue in two features: their xyloglucans lack terminal fucose residues on the side chain, and callose persists in the cell walls after the cell plates fuse with the parental plasma membrane. The lack of terminal fucose residues on xyloglucans suggests that these cell wall matrix molecules serve both structural and storage functions.     

Sub-attomole oligonucleotide and p53 cDNA determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification

Oligonucleotide (ODN)-capped gold nanoparticles (Au-NPs) were used in a sandwich assay of ODN or polynucleotide by a flow injection surface plasmon resonance (SPR). A carboxylated dextran film was immobilized onto the SPR sensor surface to eliminate nonspecific adsorption of ODN-capped Au-NPs. The tandem use of signal amplification via the adlayer of the ODN-capped Au-NPs and the differential signal detection by the bicell detector on the SPR resulted in a remarkable DNA detection level. A 39-mer target at a quantity as low as 2.1 x 10(-20)mol, corresponding to 1.38 fM in a 15 microl solution, can be measured. To our knowledge, both the concentration and quantity detection levels are the lowest among all the gene analyses conducted with SPR to this point. The method is shown to be reproducible (relative standard deviation values <16%) and to possess high sequence specificity. It is also demonstrated to be viable for sequence-specific p53 cDNA analysis. The successful elimination of nonspecific adsorption of, and the signal amplification by, ODN-capped Au-NPs renders the SPR attractive for cases where the DNA concentration is extremely low and the sample availability is severely limited.     

Solution structure and backbone dynamics of the DNA-binding domain of mouse Sox-5

The fold of the murine Sox-5 (mSox-5) HMG box in free solution has been determined by multidimensional NMR using (15)N-labeled protein and has been found to adopt the characteristic twisted L-shape made up of two wings: the major wing comprising helix 1 (F10--F25) and helix 2 (N32--A43), the minor wing comprising helix 3 (P51--Y67) in weak antiparallel association with the N-terminal extended segment. (15)N relaxation measurements show considerable mobility (reduced order parameter, S(2)) in the minor wing that increases toward the amino and carboxy termini of the chain. The mobility of residues C-terminal to Q62 is significantly greater than the equivalent residues of non-sequence-specific boxes, and these residues show a weaker association with the extended N-terminal segment than in non-sequence boxes. Comparison with previously determined structures of HMG boxes both in free solution and complexed with DNA shows close similarity in the packing of the hydrophobic cores and the relative disposition of the three helices. Only in hSRY/DNA does the arrangement of aromatic sidechains differ significantly from that of mSox-5, and only in rHMG1 box 1 bound to cisplatinated DNA does helix 1 have no kink. Helix 3 in mSox-5 is terminated by P68, a conserved residue in DNA sequence-specific HMG boxes, which results in the chain turning through approximately 90 degrees.     

The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts

It has previously been shown by immunofluorescence experiments that the cross-linking of HLA antigens into patches (by antibody reagents directed to human β₂-microglobulin) on the surfaces of cultured human fibroblasts leads to the lining up of the patches over the actomyosin-containing stress fibers lying immediately under the surface membrane. These experiments have now been extended to the resolution of the electron microscope by the use of ferritin-conjugated antibody. The results show that a substantial part of the HLA surface clusters that form by 5 min after the addition of the antibody reagents is found in small uncoated surface invaginations which are subsequently endocytosed and ultimately fuse with lysosomal bodies. At no stage in this process is there any indication that coated pits or coated vesicles participate. These and other results suggest, therefore, that there are at least two distinct mechanisms for the ligand-induced endocytosis and lysosomal processing of membrane components, one involving coated pits and the other the noncoated invaginations described in this paper. Transmembrane associations of clusters with intracellular actomyosin-containing structures may have a role in the endocytosis of these noncoated invaginations.     

Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target DNA while Au-NPs modified with oligonucleotide detection sequences played a role in recognition and signal production. Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures in solutions of relatively low salt concentration, hybridization efficiency in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which could easily and quantitatively measured by low-cost UV-vis spectrophotometric analysis. The results indicated this to be a very simple and economic strategy for detection of single-mismatched DNA strands.     

Au-NPs enhanced SPR biosensor based on hairpin DNA without the effect of nonspecific adsorption

Gold nanoparticles (Au-NPs) are usually used to amplify surface plasmon resonance (SPR) signals, however, the serious nonspecific adsorption has largely limited their practical applications. Here, we developed a novel Au-NPs enhanced biosensor without the effect of nonspecific adsorption: cutting Au-NPs off from the biosensor surface by RsaI endonuclease. In order to further improve the sensitivity, the probe DNA was designed specially. After the cleavage reaction, the residual probe DNA formed hairpin structure, which also resulted in a great change in SPR dip shift. Then, with the coaction of Au-NPs and conformation change of probe DNA, the SPR signal was amplified greatly. Using this method, we monitored the process of DNA cleavage in real-time and achieved a detection level of 5×10(-8) M. Moreover, the result of X-ray photoelectron spectroscopy (XPS) experiment further confirmed that large nonspecific adsorption existed. However, because SPR recorded a process in which the Au-NPs were cut off, the serious nonspecific adsorption did not affect the experimental result.     

Platelet-derived growth factor: morphologic and biochemical studies of binding, internalization, and degradation

Kinetic studies of binding and internalization of 125I-platelet-derived growth factor (PDGF) demonstrate that up to 15% of membrane-associated radioactivity is internalized within 2 minutes after warming to 37 degrees C in a variety of cell types. The T 1/2 for internalization is approximately 20 minutes. The T 1/2 for the subsequent appearance of degradation products in the culture medium is between 60-90 minutes following initiation of internalization. Internalization and lysosomal association of 125I-PDGF were confirmed by EM autoradiography. Quantitative studies using PDGF adsorbed to colloidal gold (gold-PDGF) demonstrate that 17% of the cell-associated sites are along coated regions of the plasma membrane (1.0 sites/micron), while 82% are associated with noncoated membrane (0.2 sites/micron). There is a significant redistribution of the gold-PDGF complexes upon warming. Within 1-2 minutes at 37 degrees C, gold particles are found within endocytic vesicles, endosomes (0.09-0.3 micron diameter), and lysosomes (greater than 0.2 micron diameter). At this time the vesicle/endosome compartment comprises 15% of the total sites and contains 0.9 sites per micron2 of surface area. The lysosomes account for 8% of the total sites and contain 0.8 sites per micron2 of surface area. Simultaneously, there is an increase in the number of gold-PDGF binding sites within coated-pits (1.6 sites/micron, 18% of the total sites) and a decrease along noncoated regions of the membrane (0.11 sites/micron, 58% of the total sites). After 15 minutes at 37 degrees C, 26% of the total sites (1.4 sites/micron2) are highly concentrated within lysosomes, while sites in the vesicle/endosome compartment remain constant. At the same time, binding sites within coated pits decrease substantially (0.5 sites/micron, 4% of the total sites), while the number of sites along noncoated regions of the membrane remain constant. Gold-PDGF was not observed associated with the Golgi complex at any time up to 120 minutes following warming. We conclude that gold-PDGF is processed via both receptor-mediated and nonspecific endocytosis and follows an intracellular pathway comparable to that followed by some other protein ligands.     

The CD of two-chain coiled coils: experiments on tropomyosin and tropomyosin segments in the tyrosine/disulfide spectral region

CD experiments are reported for several coiled-coil species in the tyrosine/disulfide (approximately 250-350-nm) region. Intact noncross-linked tropomyosin (approximately 3 degrees C) shows a negative nonsymmetric band maximal at 280 nm. This spectrum is the sum over six tyrosines/chain, and has conformational significance, since it disappears on denaturation. Experiments on an excised coiled-coil segment, each of whose chains comprise residues 11-127 of the tropomyosin sequence and only one tyrosine (Y60), reveal that not all tyrosines are alike. The spectrum at 3 degrees C shows a small negative maximum at approximately 285 nm and a substantial, hitherto unknown, positive band at approximately 270 nm, the latter masked in the parent protein by the negative contribution from the other tyrosines. A noncross-linked coiled-coil segment comprising residues 142-281, in which Y60 is absent, shows no such positive band. This peculiarity of Y60 is confirmed by absorbance spectra, with the extinction coefficient of Y60 larger in benign media than the average of the other tyrosines. Intact (3 degrees C) C190 cross-linked tropomyosin is known to yield, besides tyrosine contributions, a positive maximum at approximately 300 nm. Subtracting the corresponding data for noncross-linked tropomyosin shows that the disulfide spectrum itself actually has two equal, partly resolved bands at, respectively, 250 and 280 nm. The existence of a chiral disulfide argues for a relatively rigid, perhaps strained, local coiled coil. A C190 cross-linked segment comprising residues 142-281 shows a chiral disulfide spectrum like tropomyosin's, but another segment, comprising residues 168-284, shows none; thus removal of residues 142-167 causes loss of chirality at C190, over 20 residues away. These spectra thus contain important information on the subtle local differences in coiled-coil structures.     

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more efficient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes.     

Visual detection of Hg²⁺ in aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes

We report a sensitive method for visual detection of mercury ions (II) (Hg2?) in aqueous solution by using gold nanoparticles (Au-NPs) and thymine (T)-rich hairpin DNA probes. The thiolated hairpin DNA probe was immobilized on the Au-NP surface through a self-assembling method. Another thymine-rich, digoxin-labeled DNA probe was introduced to form DNA duplexes on the Au-NP surface with thymine-Hg2?-thymine (T-Hg2?-T) coordination in the presence of Hg2?. The Au-NPs associated with the formed duplexes were captured on the test zone of a lateral flow strip biocomponent (LFSB) by immunoreaction events between the digoxin on the duplexes and anti-digoxin antibodies on the LFSB. The accumulation of Au-NPs produced a characteristic red band on the test zone, enabling visual detection of Hg2? without instrumentation. A detection limit of 0.1 nM was obtained under optimal experimental conditions. This method provides a simple, rapid, sensitive approach for the detection of Hg2? and shows great promise for point-of-care and in-field detection of environmentally toxic mercury.     

pH-dependent assembly of DNA-gold nanoparticles based on the i-motif

Oligonucleotides containing stretches of 2 '-deoxycytidine residues were immobilized on 15 nm gold nanoparticles. Under acidic pH conditions a reversible supramolecular assembly is formed, induced by the formation of the tetrameric i-motif structure. The replacement of 2 '-deoxycytidine by 5-propynyl-2 '-deoxycytidine (dC*) leads to novel i-motif structures. Oligonucleotides incorporating multiple residues of dC* were immobilized on 15 nm gold nanoparticles and are able to aggregate into i-motif structures even at non-optimal pH values.     

Binding, uptake, and transcytosis of ligands for mannose-specific receptors in rat liver: an electron microscopic study

We have investigated the initial distribution of mannose-specific binding sites in rat liver as well as the uptake and transcytosis pathways of ligands for this receptor in in situ and in vivo experiments. As ligands we used mannan adsorbed onto colloidal gold particles of sizes 5, 17, and 35 nm (Man-Au5, Man-Au17, or Man-Au35). The in situ binding pattern of Man-Au5 in the prefixed liver is identical to the one described earlier for galactose-exposing ligands in the same organ. With the exception of the binding by hepatocytes, where only scarce binding of Man-Au5 was observed, ligands were found adhering in a preclustered pattern all over the cell surface of liver macrophages and binding in aggregates over the coated pits of endothelial cells. In double-labeling experiments different particle sizes were used for glycoproteins with terminal mannosyl or galactosyl residues. This simultaneous localization of the two binding activities revealed that on endothelial cells the two activities are always found to be present in the same coated pit. On liver macrophages the clustered binding occurred at different membrane areas. Uptake and transcytosis of Man-Au5, 17, 35 were studied after their injection into the tail vein. Three and fifteen minutes after injection most of the Man-Au5 and all of Man-Au17 or Man-Au35 was found in sinusoidal liver cells, i.e., macrophages and endothelial cells. One hour after injection, endocytosed ligand is redistributed from large--presumably lysosomal--vacuoles to small noncoated vesicles that are localized predominantly near the space of Dissé. Between 1 and 40 h after injection, ligands of all sizes are transcytosed and found in the hepatocytes. No ligand accumulation is observed in hepatocytes as an indirect indication for secretion into bile. With this investigation we give evidence for transcytotic activity not only of liver endothelium but also of the resident liver macrophages.     

Circulation and distribution of gold nanoparticles and induced alterations of tissue morphology at intravenous particle delivery

Kinetics, biodistribution, and histological studies were performed to evaluate the particle‐size effects on the distribution of 15 nm and 50 nm PEG‐coated colloidal gold (CG) particles and 160 nm silica/gold nanoshells (NSs) in rats and rabbits. The above nanoparticles (NPs) were used as a model because of their importance for current biomedical applications such as photothermal therapy, optical coherence tomography, and resonance‐scattering imaging. The dynamics of NPs circulation in vivo was evaluated after intravenous administration of 15 nm CG NPs to rabbit, and the maximal concentrations of gold were observed 15–30 min after injection. Rats were injected in the tail vein with PEG‐coated NPs (about 0.3 mg Au/kg rats). 24 h after injection, the accumulation of gold in different organs and blood was determined by atomic absorption spectroscopy. In accordance with the published reports, we observed 15 nm particles in all organs with rather smooth distribution over liver, spleen and blood. By contrast, the larger NSs were accumulated mainly in the liver and spleen. For rabbits, the biodistribution was similar (72 h after intravenous injection). We report also preliminary data on the light microscopy and TEM histological examination that allows evaluation of the changes in biotissues after gold NPs treatment. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors

Monomeric ferritin-insulin and high-resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3-L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37% C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in noncoated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3-L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue-specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.