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1.
Nature ; 559(7712): 135-139, 2018 07.
Article in English | MEDLINE | ID: mdl-29950717

ABSTRACT

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.


Subject(s)
Cryoelectron Microscopy , Plasmodium vivax/chemistry , Plasmodium vivax/ultrastructure , Protozoan Proteins/chemistry , Protozoan Proteins/ultrastructure , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, CD/ultrastructure , Binding Sites , Humans , Malaria Vaccines/immunology , Models, Molecular , Mutation , Plasmodium vivax/cytology , Plasmodium vivax/genetics , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Receptors, Transferrin/chemistry , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Receptors, Transferrin/ultrastructure , Reticulocytes/metabolism , Structure-Activity Relationship , Transferrin/chemistry , Transferrin/metabolism , Transferrin/ultrastructure
2.
Immunol Lett ; 140(1-2): 44-51, 2011 Oct 30.
Article in English | MEDLINE | ID: mdl-21704079

ABSTRACT

Nano-spatial distribution of cell surface molecules on cell membrane fluctuations during T-cell activation has not been reported. In this study, we innovated application of near-field scanning optical microscopy (NSOM)/quantum dots (QDs)-based nanotechnology through three-dimensional image fusion algorithm to merge the simultaneously obtained dual-color fluorescence information and three-dimensional topography. This novel imaging system made it possible to visualize nano-spatial distribution and organization of early-activation molecules CD69 and late-activation molecules CD71 on cell-membrane fluctuations during T-cell activation. Interestingly, most CD69 molecules were clustered to form 250-500nm nano-domains polarizing predominantly in the peak of the cell-membrane fluctuations. In contrast, although CD71 molecules were also clustered as 250-500nm nano-domains, they polarized dominantly in the valley of the cell-membrane fluctuations. The peak-valley polarities of CD69 nano-domains and CD71 nano-domains implied their different functions. CD69 nano-domains polarizing on membrane-peak fluctuations might serve as transient platforms driving TCR/CD3-induced signaling and activation, whereas CD71 nano-domains distributing in the membrane-valley fluctuations appeared to facilitate iron uptake for increased metabolisms in T-cell activation. Importantly, this NSOM/QD-based fluorescence-topographic image fusion provides a powerful tool to visualize nano-spatial distribution of cell-surface molecules on cell-membrane fluctuations and enable better understanding of distribution-function relationship.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Cell Surface Extensions/metabolism , Imaging, Three-Dimensional , Lectins, C-Type/metabolism , Receptors, Transferrin/metabolism , T-Lymphocytes/metabolism , Animals , Antigens, CD/ultrastructure , Antigens, Differentiation, T-Lymphocyte/ultrastructure , Cell Polarity/immunology , Cell Surface Extensions/ultrastructure , Fluorescent Antibody Technique, Indirect , Humans , Lectins, C-Type/ultrastructure , Lymphocyte Activation , Macaca mulatta , Membrane Microdomains/ultrastructure , Microscopy, Confocal , Nanotechnology , Quantum Dots , Receptor Aggregation/immunology , Receptors, Transferrin/ultrastructure , T-Lymphocytes/ultrastructure , Time Factors
3.
Structure ; 18(1): 17-27, 2010 Jan 13.
Article in English | MEDLINE | ID: mdl-20152149

ABSTRACT

Single particle reconstruction from cryoelectron microscopy images, though emerging as a powerful means in structural biology, is faced with challenges as applied to asymmetric proteins smaller than megadaltons due to low contrast. Zernike phase plate can improve the contrast by restoring the microscope contrast transfer function. Here, by exploiting simulated Zernike and conventional defocused cryoelectron microscope images with noise characteristics comparable to those of experimental data, we quantified the efficiencies of the steps in single particle analysis of ice-embedded RNA polymerase II (500 kDa), transferrin receptor complex (290 kDa), and T7 RNA polymerase lysozyme (100 kDa). Our results show Zernike phase plate imaging is more effective as to particle identification and also sorting of orientations, conformations, and compositions. Moreover, our analysis on image alignment indicates that Zernike phase plate can, in principle, reduce the number of particles required to attain near atomic resolution by 10-100 fold for proteins between 100 kDa and 500 kDa.


Subject(s)
Cryoelectron Microscopy/methods , Muramidase/analysis , RNA Polymerase II/analysis , Receptors, Transferrin/analysis , Bacteriophage T7 , Muramidase/ultrastructure , RNA Polymerase II/ultrastructure , Receptors, Transferrin/ultrastructure , Saccharomyces cerevisiae
4.
J Electron Microsc (Tokyo) ; 59(1): 53-63, 2010.
Article in English | MEDLINE | ID: mdl-19643814

ABSTRACT

We evaluated imaging plates (IPs) and the DITABIS Micron scanner for their use in recording images of negatively stained single-particle specimens and electron diffraction patterns of two-dimensional crystals. We first established the optimal imaging and read-out conditions for images of negatively stained single-particle specimens using the signal-to-noise ratio of the images as the evaluation criterion. We found that images were best recorded on IPs at a magnification of 67,000x, read out with a gain setting of 20,000 and a laser power setting of 30% with subsequent binning over 2 x 2 pixels. Our results show that for images of negatively stained specimens, for which the resolution is limited to approximately 20 A, IPs are a good alternative to EM film. We also compared IPs with a 2K x 2K Gatan charge-coupled device (CCD) camera for their use in recording electron diffraction patterns of sugar-embedded two-dimensional crystals. Diffraction patterns of aquaporin-0 recorded on IPs and with the CCD camera showed reflections beyond 3 A and had similar R(Friedel) as well as R(merge) values. IPs can thus be used to collect diffraction patterns, but CCD cameras are more convenient and remain the best option for recording electron diffraction patterns.


Subject(s)
Image Processing, Computer-Assisted/instrumentation , Microscopy, Electron, Transmission/instrumentation , Aquaporins/ultrastructure , Crystallography/instrumentation , Crystallography/methods , Eye Proteins/ultrastructure , Humans , Microscopy, Electron, Transmission/methods , Multiprotein Complexes/ultrastructure , Photography , Proteasome Endopeptidase Complex/ultrastructure , Receptors, Transferrin/ultrastructure , Transferrin/ultrastructure
5.
J Mol Biol ; 382(2): 423-33, 2008 Oct 03.
Article in English | MEDLINE | ID: mdl-18655791

ABSTRACT

We have recently developed monolayer purification as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single-particle electron microscopy (EM) without biochemical purification. Here, we introduce the Affinity Grid, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomal complexes from Escherichia coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50 S subunit rplC. Ribosomal complexes with or without associated mRNA could be prepared depending on the way the sample was applied to the Affinity Grid . Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50 S ribosomal subunit as well as the 70 S ribosome and 30 S ribosomal subunit from images of the same sample. We established that Affinity Grids are stable for some time in the presence of glycerol and detergents, which allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilized membrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single-particle EM specimens of soluble complexes and membrane proteins.


Subject(s)
Lipids/chemistry , Macromolecular Substances/isolation & purification , Microscopy, Electron/instrumentation , Aquaporins/isolation & purification , Humans , Microscopy, Electron/methods , Models, Molecular , Protein Conformation , Protein Subunits/isolation & purification , Receptors, Transferrin/isolation & purification , Receptors, Transferrin/ultrastructure , Ribosomal Protein L3 , Ribosomes/chemistry , Ribosomes/ultrastructure , Transferrin/isolation & purification , Transferrin/ultrastructure
6.
Proc Natl Acad Sci U S A ; 105(12): 4703-8, 2008 Mar 25.
Article in English | MEDLINE | ID: mdl-18347330

ABSTRACT

Visualizing macromolecular complexes by single-particle electron microscopy (EM) entails stringent biochemical purification, specimen preparation, low-dose imaging, and 3D image reconstruction. Here, we introduce the "monolayer purification" method, which employs nickel-nitrilotriacetic acid (Ni-NTA) functionalized lipids for simultaneously purifying His-tagged complexes directly from cell lysates while producing specimens suitable for single-particle EM. The method was established by using monolayers containing Ni-NTA lipid to specifically adsorb His-tagged transferrin-transferrin receptor (Tf-TfR) complexes from insect and mammalian cell extracts. The specificity and sensitivity of the method could be improved by adding imidazole to the extracts. The monolayer-purified Tf-TfR samples could be vitrified and used to calculate a 3D reconstruction of the complex. Monolayer purification was then used to rapidly isolate ribosomal complexes from bacteria by overexpressing a single His-tagged ribosomal subunit. The resulting monolayer samples allowed calculation of a cryo-EM 3D reconstruction of the Escherichia coli 50S ribosomal subunit.


Subject(s)
Chemistry Techniques, Analytical/methods , Cryoelectron Microscopy/methods , Multiprotein Complexes/isolation & purification , Multiprotein Complexes/ultrastructure , Adsorption , Animals , Cell Extracts , Histidine/metabolism , Lipid Metabolism , Nickel , Nitrilotriacetic Acid/metabolism , Oligopeptides/metabolism , Receptors, Transferrin/metabolism , Receptors, Transferrin/ultrastructure , Ribosomal Protein L3 , Ribosomal Proteins/metabolism , Ribosomes/ultrastructure , Transferrin/metabolism , Transferrin/ultrastructure
7.
Biophys J ; 94(1): 230-40, 2008 Jan 01.
Article in English | MEDLINE | ID: mdl-17872962

ABSTRACT

Interaction between the iron transporter protein transferrin (Tf) and its receptor at the cell surface is fundamental for most living organisms. Tf receptor (TfR) binds iron-loaded Tf (holo-Tf) and transports it to endosomes, where acidic pH favors iron release. Iron-free Tf (apo-Tf) is then brought back to the cell surface and dissociates from TfR. Here we investigated the Tf-TfR interaction at the single-molecule level under different conditions encountered during the Tf cycle. An atomic force microscope tip functionalized with holo-Tf or apo-Tf was used to probe TfR. We tested both purified TfR anchored to a mica substrate and in situ TfR at the surface of living cells. Dynamic force measurements showed similar results for TfR on mica or at the cell surface but revealed striking differences between holo-Tf-TfR and apo-Tf-TfR interactions. First, the forces necessary to unbind holo-Tf and TfR are always stronger compared to the apo-Tf-TfR interaction. Second, dissociation of holo-Tf-TfR complex involves overcoming two energy barriers, whereas the apo-Tf-TfR unbinding pathway comprises only one energy barrier. These results agree with a model that proposes differences in the contact points between holo-Tf-TfR and apo-Tf-TfR interactions.


Subject(s)
Microscopy, Atomic Force/methods , Models, Chemical , Models, Molecular , Receptors, Transferrin/chemistry , Receptors, Transferrin/ultrastructure , Transferrin/chemistry , Transferrin/ultrastructure , Binding Sites , Computer Simulation , Protein Binding , Protein Conformation , Protein Interaction Mapping
8.
Mol Biol Cell ; 18(6): 2226-43, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17409357

ABSTRACT

Previously, FRET confocal microscopy has shown that polymeric IgA-receptor (pIgA-R) is distributed in a clustered manner in apical endosomes. To test whether different membrane-bound components form clusters during membrane trafficking, live-cell quantitative FRET was used to characterize the organization of pIgA-R and transferrin receptor (TFR) in endocytic membranes of polarized MDCK cells upon internalization of donor- and acceptor-labeled ligands. We show that pIgA-R and TFR complexes form increasingly organized clusters during cotransport from basolateral to perinuclear endosomes. The organization of these receptor clusters in basolateral versus perinuclear/apical endosomes is significantly different; the former showing a mixed random/clustered distribution while the latter highly organized clusters. Our results indicate that although both perinuclear and apical endosomes comprise pIgA-R and TFR clusters, their E% levels are significantly different suggesting that these receptors are packed into clusters in a distinct manner. The quantitative FRET-based assay presented here suggests that different receptor complexes form clusters, with diverse levels of organization, while being cotransported via the polarized endocytic pathways.


Subject(s)
Endocytosis/physiology , Immunoglobulin A/metabolism , Receptors, Polymeric Immunoglobulin/metabolism , Receptors, Transferrin/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Cell Polarity , Cell Shape , Dogs , Endosomes/metabolism , Endosomes/ultrastructure , Fluorescence Resonance Energy Transfer , Humans , Models, Biological , Receptors, Polymeric Immunoglobulin/genetics , Receptors, Polymeric Immunoglobulin/ultrastructure , Receptors, Transferrin/ultrastructure
10.
J Mol Biol ; 355(5): 1048-65, 2006 Feb 03.
Article in English | MEDLINE | ID: mdl-16343539

ABSTRACT

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.


Subject(s)
Imaging, Three-Dimensional , Protein Conformation , Receptors, Transferrin/chemistry , Staining and Labeling/methods , Transferrin/chemistry , Carbon/chemistry , Glucose/chemistry , Glycerol/chemistry , Humans , Microscopy, Electron/methods , Models, Molecular , Molybdenum/chemistry , Organometallic Compounds/chemistry , Receptors, Transferrin/ultrastructure , Software , Transferrin/ultrastructure
11.
J Struct Biol ; 152(3): 204-10, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16343946

ABSTRACT

Most organisms depend on iron as a co-factor for proteins catalyzing redox reactions. Iron is, however, a difficult element for cells to deal with, as it is insoluble in its ferric (Fe3+) form and potentially toxic in its ferrous (Fe2+) form. Thus, in vertebrates iron is transported through the circulation bound to transferrin (Tf) and delivered to cells through an endocytotic cycle involving the transferrin receptor (TfR). We have previously presented a model for the Tf-TfR complex in its iron-bearing form, the diferric transferrin (dTf)-TfR complex [Cheng, Y., Zak, O., Aisen, P., Harrison, S.C., Walz, T., 2004. Structure of the human transferrin receptor-transferrin complex. Cell 116, 565-576]. We have now calculated a single particle reconstruction for the complex in its iron-free form, the apo-transferrin (apoTf)-TfR complex. The same density map was obtained by aligning raw particle images or class averages of the vitrified apoTf-TfR complex to reference models derived from the structures of the dTf-TfR or apoTf-TfR complex. We were unable to improve the resolution of the apoTf-TfR density map beyond 16A, most likely because of significant structural variability of Tf in its iron-free state. The density map does, however, support the model for the apoTf-TfR we previously proposed based on the dTf-TfR complex structure, and it suggests that receptor-bound apoTf prefers to adopt an open conformation.


Subject(s)
Apoproteins/ultrastructure , Cryoelectron Microscopy , Imaging, Three-Dimensional , Receptors, Transferrin/ultrastructure , Transferrin/ultrastructure , Apoproteins/chemistry , Apoproteins/metabolism , Humans , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/ultrastructure , Protein Binding , Receptors, Transferrin/chemistry , Receptors, Transferrin/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Transferrin/chemistry , Transferrin/metabolism
12.
Mol Cell Proteomics ; 4(12): 1959-67, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16332734

ABSTRACT

The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.


Subject(s)
Receptors, Transferrin/chemistry , Receptors, Transferrin/metabolism , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Cryoelectron Microscopy , Humans , Iron/metabolism , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Protein Conformation , Receptors, Transferrin/ultrastructure
13.
Biophys J ; 88(3): 2266-77, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15613635

ABSTRACT

Molecules undergo non-Brownian diffusion in the plasma membrane, but the mechanism behind this anomalous diffusion is controversial. To characterize the anomalous diffusion in the complex system of the plasma membrane and to understand its underlying mechanism, single-molecule/particle methods that allow researchers to avoid ensemble averaging have turned out to be highly effective. However, the intrinsic problems of time-averaging (resolution) and the frequency of the observations have not been explored. These would not matter for the observations of simple Brownian particles, but they do strongly affect the observation of molecules undergoing anomalous diffusion. We examined these effects on the apparent motion of molecules undergoing simple, totally confined, or hop diffusion, using Monte Carlo simulations of particles undergoing short-term confined diffusion within a compartment and long-term hop diffusion between these compartments, explicitly including the effects of time-averaging during a single frame of the camera (exposure time) and the frequency of observations (frame rate). The intricate relationships of these time-related experimental parameters with the intrinsic diffusion parameters have been clarified, which indicated that by systematically varying the frame time and rate, the anomalous diffusion can be clearly detected and characterized. Based on these results, single-particle tracking of transferrin receptor in the plasma membrane of live PtK2 cells were carried out, varying the frame time between 0.025 and 33 ms (0.03-40 kHz), which revealed the hop diffusion of the receptor between 47-nm (average) compartments with an average residency time of 1.7 ms, with the aid of single fluorescent-molecule video imaging.


Subject(s)
Cell Membrane/chemistry , Cell Membrane/metabolism , Kidney/chemistry , Kidney/metabolism , Models, Biological , Protein Transport/physiology , Receptors, Transferrin/chemistry , Receptors, Transferrin/metabolism , Animals , Cell Membrane/ultrastructure , Computer Simulation , Diffusion , Dipodomys , Kidney/ultrastructure , Microscopy, Fluorescence , Microscopy, Video , Models, Chemical , Receptors, Transferrin/ultrastructure
14.
EMBO J ; 22(14): 3591-601, 2003 Jul 15.
Article in English | MEDLINE | ID: mdl-12853474

ABSTRACT

The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid 'raft' domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23-107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.


Subject(s)
Endocytosis , Glycosylphosphatidylinositols/metabolism , Neurons, Afferent/metabolism , PrPC Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cells, Cultured , Coated Pits, Cell-Membrane/metabolism , Coated Pits, Cell-Membrane/ultrastructure , Disulfides/chemistry , Kinetics , Membrane Microdomains/metabolism , Mice , Molecular Sequence Data , Neuroblastoma/metabolism , Neuroblastoma/pathology , Neuroblastoma/ultrastructure , Neurons, Afferent/cytology , Neurons, Afferent/ultrastructure , PrPC Proteins/chemistry , PrPC Proteins/genetics , PrPC Proteins/ultrastructure , Protein Binding , Protein Structure, Tertiary , Receptors, Transferrin/metabolism , Receptors, Transferrin/ultrastructure , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/ultrastructure , Thy-1 Antigens/metabolism , Thy-1 Antigens/ultrastructure
15.
Neoplasia ; 5(6): 495-506, 2003.
Article in English | MEDLINE | ID: mdl-14965443

ABSTRACT

Noninvasive imaging of differences between the molecular properties of cancer and normal tissue has the potential to enhance the detection of tumors. Because overexpression of endogenous transferrin receptor (TfR) has been qualitatively described for various cancers and is presumably due to malignant transformation of cells, TfR may represent a suitable target for application of molecular imaging technologies to increase detection of smaller tumors. In the work reported here, investigation into the biology of this receptor using electron microscopy has demonstrated that iron oxide particles targeted to TfR are internalized and accumulate in lysosomal vesicles within cells. Biochemical analysis of the interaction of imaging probes with cells overexpressing the TfR demonstrated that the extent of accumulation, and therefore probe efficacy, is dependent on the nature of the chemical cross-link between transferrin and the iron oxide particle. These data were utilized to design and synthesize an improved imaging probe. Experiments demonstrate that the novel magnetic resonance imaging (MRI) probe is sensitive enough to detect small differences in endogenous TfR expression in human cancer cell lines. Quantitative measurement of TfR overexpression in a panel of 27 human breast cancer patients demonstrated that 74% of patient cancer tissues overexpressed the TfR and that the sensitivity of the new imaging agent was suitable to detect TfR overexpression in greater than 40% of these cases. Based on a biochemical and cell biological approach, these studies have resulted in the synthesis and development of an improved MRI probe with the best in vitro and in vivo imaging properties reported to date.


Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/metabolism , Carcinoma, Intraductal, Noninfiltrating/metabolism , Carcinoma/metabolism , Molecular Probes/chemical synthesis , Receptors, Transferrin/metabolism , Animals , Blotting, Western , Breast Neoplasms/diagnosis , Carcinoma/diagnosis , Carcinoma, Intraductal, Noninfiltrating/diagnosis , Diagnostic Imaging , Ferric Compounds/chemistry , Ferric Compounds/metabolism , Humans , Lasers , Magnetic Resonance Imaging , Microdissection , Microscopy, Electron , Receptors, Transferrin/chemistry , Receptors, Transferrin/ultrastructure , Reverse Transcriptase Polymerase Chain Reaction , Succinimides/chemistry , Transfection , Tumor Cells, Cultured
16.
Structure ; 6(10): 1235-43, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9782058

ABSTRACT

BACKGROUND: The transferrin receptor (TfR) regulates the cellular uptake of serum iron. Although the TfR serves as a model system for endocytosis receptors, neither crystal structure analysis nor electron microscopy has yet revealed the molecular dimensions of the TfR. To derive the first molecular model, we analyzed purified, lipid-reconstituted human TfR by high-resolution electron microscopy. RESULTS: A structural model of phospholipid-reconstituted TfR was derived from 72 cryo-electron microscopic images. The TfR dimer consists of a large extracellular globular domain (6.4 x 7.5 x 10.5 nm) separated from the membrane by a thin molecular stalk (2.9 nm). A comparative protein sequence analysis suggests that the stalk corresponds to amino acid residues 89-126. Under phospholipid-reconstitution conditions, the human TfR not only integrates into vesicles, but also forms rosette-like structures called proteoparticles. Scanning transmission electron microscopy revealed an overall diameter of 31.5 nm and a molecular mass of 1669 +/- 26 kDa for the proteoparticles, corresponding to nine TfR dimers. The average mass of a single receptor dimer was determined as being 186 +/- 4 kDa. CONCLUSIONS: Proteoparticles resemble TfR exosomes that are expelled by sheep reticulocytes upon maturation. The structure of proteoparticles in vitro is thus interpreted as being the result of the TfR's strong self-association potential, which might facilitate the endosomal sequestration of the TfR away from other membrane proteins and its subsequent return to the cell surface within tubular structures. The stalk is assumed to facilitate the tight packing of receptor molecules in coated pits and recycling tubuli.


Subject(s)
Phospholipids/chemistry , Receptors, Transferrin/ultrastructure , Animals , Chickens , Cricetinae , Cryoelectron Microscopy , Humans , Mice , Microscopy, Electron, Scanning Transmission , Models, Structural , Phosphatidylcholines/chemistry , Receptors, Transferrin/chemistry
17.
J Cell Biol ; 131(6 Pt 1): 1387-401, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8522599

ABSTRACT

The transfer of newly synthesized membrane proteins moving from the rough endoplasmic reticulum (RER) to the Golgi complex has been studied by electron microscopy in HEp-2 cells transfected with cDNAs for chimeric proteins. These proteins consist of a reporter enzyme, horseradish peroxidase (HRP), anchored to the transmembrane domains of two integral membrane proteins, the transferrin receptor and sialyl-transferase. The chimeras are distributed throughout the nuclear envelope, RER, vesicular tubular clusters (VTCs) and a network of tubules in the cis-Golgi area. At 20 degrees C tubules containing chimera connect the RER to the VTCs and to the cis-Golgi network. On transfer to 37 degrees C in the presence of dithiothreitol (DTT), the chimeras are seen to move from the RER and through the Golgi stack. With this temperature shift the direct connections with the RER are lost and free vesicles form; some of these vesicles contain HRP reaction product which is much more concentrated than in the adjacent RER while others lack reaction product entirely. In cells expressing SSHRPKDEL, DAB reaction product remains distributed throughout the RER, the VTCs, and the cis-Golgi network for prolonged periods in the presence of DTT and almost all of the vesicles which form at 37 degrees C are DAB-positive. Together these observations demonstrate that all three chimeras are transported from the RER to the cis-Golgi in free, 40-60-nm vesicles at 37 degrees C. They also suggest that the retrograde traffic which carries SSHRPKDEL back to the RER is probably mediated by vesicles with a similar morphology but which, in cells expressing membrane-anchored chimeras, lack detectable reaction product.


Subject(s)
Endoplasmic Reticulum, Rough/metabolism , Golgi Apparatus/metabolism , Protein Sorting Signals , Amino Acid Sequence , Base Sequence , Biological Transport/physiology , Cell Compartmentation/physiology , Dithiothreitol/pharmacology , Endoplasmic Reticulum, Rough/ultrastructure , Exocytosis/physiology , Golgi Apparatus/ultrastructure , Horseradish Peroxidase , Humans , Laryngeal Neoplasms , Membrane Proteins/metabolism , Membrane Proteins/ultrastructure , Microscopy, Electron , Molecular Sequence Data , Oligopeptides/metabolism , Receptors, Transferrin/biosynthesis , Receptors, Transferrin/metabolism , Receptors, Transferrin/ultrastructure , Recombinant Fusion Proteins/metabolism , Sialyltransferases/metabolism , Temperature , Tumor Cells, Cultured/enzymology , p-Dimethylaminoazobenzene
18.
Biochemistry ; 34(18): 6196-207, 1995 May 09.
Article in English | MEDLINE | ID: mdl-7742325

ABSTRACT

We have reconstituted the human placental transferrin receptor (hTfR) into phospholipid vesicles using either dialysis, dilution, or gel filtration. Several different detergents and phospholipids and a variety of lipid-to-protein weight ratios were tested. Preformed vesicles as well as detergent-solubilized phospholipids were used. Reconstituted mixtures were fractionated by sucrose density gradient centrifugation and screened for ferritransferrin binding activity, and peak fractions were analyzed by electron microscopy. The efficiency of reconstitution was strongly influenced by the choice of the phospholipids used and the reconstitution method. Best results were obtained when hTfR was dissolved with octylpolyoxyethylene, mixed with detergent-solubilized soy bean lecithin, and reconstituted by slow dialysis. The data show that hTfR capable of binding ferritransferrin was reconstituted into vesicles with an irregular surface and many protrusions. In addition the reconstitution of hTfR resulted in the formation of tubular structures proceeding from the vesicle surface, which may serve as an in vitro model for future studies on the relevance of self-assembly processes for cellular endocytosis.


Subject(s)
Lipid Bilayers/metabolism , Receptors, Transferrin/metabolism , Centrifugation, Density Gradient , Female , Humans , Lipid Bilayers/chemistry , Microscopy, Electron , Placenta/metabolism , Pregnancy , Receptors, Transferrin/ultrastructure , Transferrin/metabolism
19.
EMBO J ; 12(4): 1713-21, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8467813

ABSTRACT

Wild-type human transferrin receptor (hTfR), like endogenous canine receptor, is expressed almost exclusively (97%) at the basolateral membrane of transfected Madin-Darbey canine kidney (MDCK) cells. We investigated the role of two distinct features of the hTfR cytoplasmic domain, namely the endocytic signal and the unique phosphorylation site, in polarized cell surface delivery. Basolateral location was not altered by point mutation of Ser24-->Ala24, indicating that phosphorylation is not involved in vectorial sorting of hTfR. The steady state distribution of hTfR was partially affected by a deletion of 36 cytoplasmic residues encompassing the internalization sequence. However, 80% of the receptors were still basolateral. As assessed by pulse-chase experiments in combination with biotinylation, newly synthesized wild-type and deletion mutant receptors were directly sorted to the domain of their steady state residency. Although both receptors could bind human transferrin, endocytosis of the deletion mutant was strongly impaired at either surface. These data indicate that the predominant basolateral targeting signal of hTfR is independent of the internalization sequence.


Subject(s)
Receptors, Transferrin/physiology , Transferrin/metabolism , Amino Acid Sequence , Animals , Cell Compartmentation , Cell Line , Cell Polarity , Cytoplasm/ultrastructure , DNA Mutational Analysis , Dogs , Endocytosis , Humans , In Vitro Techniques , Microscopy, Electron , Molecular Sequence Data , Phosphoproteins/physiology , Phosphoproteins/ultrastructure , Phosphorylation , Receptors, Transferrin/ultrastructure , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Sequence Deletion , Structure-Activity Relationship , Transfection
20.
Biosci Rep ; 12(6): 471-82, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1298437

ABSTRACT

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.


Subject(s)
Placenta/metabolism , Receptors, Transferrin/isolation & purification , Cell Membrane/metabolism , Chromatography, Affinity , Female , Humans , In Vitro Techniques , Liposomes , Microscopy, Electron , Octoxynol , Polyethylene Glycols , Pregnancy , Protein Conformation , Receptors, Transferrin/chemistry , Receptors, Transferrin/ultrastructure , Trypsin
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