Updated: December 5, 2003
N A N O P R O B E S E - N E W S
Vol. 4, No. 12 December 5, 2003
This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.
Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing tech@nanoprobes.com.
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The high penetration and density of labeling with Nanogold-Fab', and uniformity of the gold enhancement process, make it ideal for complex morphological studies. Polishchuk and group continue their investigation of the mechanisms of Golgi function using Nanogold® with gold enhancement, correlated with fluorescence confocal microscopy, as one of a cluster of morphological techniques to study the formation of Golgi-to-plasma membrane (PM) carriers (GPCs) and how these large structures emerge from the trans-Golgi network (TGN). Green fluorescent protein (GFP)-labeled cargo proteins were used to show that GPCs form by cleavage from 1- to 8-micrometer-long membrane precursors which are extruded from the Golgi complex along microtubules (MTs) by kinesin. These precursors were then investigated by electron microscopy.
This group has developed a procedure for correlative fluorescence and electron microscopy in which cells are first transfected with a GFP fusion protein, observed by confocal or inverted fluorescence microscopy with computerized image acquisition, then fixed while this acquisition is in progress so that the electron microscopic images may be correlated exactly with the fluorescent images of dynamic processes. For this experiment, Cos7 cells transfected with Vesicular Stomatitis Virus (VSV) were used for immuno-EM identification of TGN exit sites of VSVG-GFP. After visualization of VSVG-GFPpositive exit sites by time-lapse confocal microscopy, the following procedure was used for electron microscopy:
- Fix cells while still acquiring confocal images, using first 0.1 % glutaraldehyde and 4 % paraformaldehyde in 0.02 M HEPES buffer, pH 7.4 (10 minutes), then 4 % paraformaldehyde in the same buffer (30 minutes).
- Wash 5 minutes with 2 mL of PBS
- Incubate 30 minutes with 2 mL of blocking solution (100 mL of PBS containing 0.5 g BSA, 0.1 g saponin, and 0.27 g ammonium chloride)
- Incubate overnight with primary antibody diluted in the blocking solution; in this case polyclonal antibody (pAb) against the lumenal domain of VSVG (K. Simons, Max Planck Institute, Dresden, Germany). Dilutions vary with antibody, but should be 5 - 10 X those used for immunofluorescence.
- Wash 6 X 2 minutes with 2 mL PBS.
- Dilute Nanogold-conjugated Fab' secondary antibody 1:50 (v/v) in blocking solution and add to the cells; incubate 2 hours.
- Wash 6 X 2 minutes with 2 mL PBS.
- Fix cells 5 minutes with 1 mL of 1% glutaraldehyde in 0.2 M HEPES buffer, pH 7.3, then rinse three times with distilled water.
- Develop with GoldEnhance EM for 6 - 10 minutes.
Cells were then embedded in Epon-812 and cut in 40-60nm serial sections, or cryosections were prepared as described previously (Bonfanti et al, 1998). EM images were acquired from thin sections under a Philips Tecnai-12 electron microscope by using an ULTRA VIEW charge-coupled device digital camera. Thin sections of the specimens taken from 20 and 32°C (release of VSVG for 10 min) were used for quantification of gold labeling density of VSVG at the trans-Golgi (TG)-TGN and on GPCs.
When membrane (vesicle) fusion is inhibited, bona fide GPC precursors and GPCs continue forming with normal dynamics and structure, indicating that they do not arise from the fusion of smaller vesicles. Both small (G protein of vesicular stomatitis virus; VSVG) and supramolecular (procollagen-I; PC-I) cargo proteins were shown to leave the Golgi within the same GPCs, suggesting that both cargoes use the same mechanism to exit the TGN. These observations all agree in indicating that the process of GPC formation occurs by direct extrusion of large portions of the TGN. GPC formation is neither coupled to the concentration of cargo nor associated with the recruitment of known adaptor and coat proteins to the GPC precursors budding from the TGN. From these results, it was inferred that exit from the TGN of a constitutive traffic marker, the VSVG protein, occurs by bulk flow via a three-step process: (1) formation of a tubular-reticular TGN domain (GPC precursor) that includes PM-directed proteins and excludes other cargo and Golgi-resident proteins; (2) docking of this preformed domain on microtubules and its kinesin-mediated extrusion; and finally (3) the detachment of the extruded domain by membrane fission. Export from the Golgi therefore occurs via the formation, protrusion and en bloc cleavage of specialized TGN tubular-saccular domains.
Reference:
Polishchuk, E. V.; Di Pentima, A.; Luini, A., and Polishchuk, R. S.: Mechanism of Constitutive Export from the Golgi: Bulk Flow via the Formation, Protrusion, and En Bloc Cleavage of large trans-Golgi Network Tubular Domains. Mol. Biol. Cell, 14, 4470-4485 (2003).
This group used the same method previously to localize RNA polymerase I (RPI) in the same cell line for a study to localize transcribing rRNA genes at the ultrastructural level and described their three-dimensional organization within the nucleolus by electron tomography.
The procedure for correlative video light and electron microscopy is described and discussed in the following reference:
Reference:
Polishchuk, R. S., and Mironov, A. A.: Correlative video/light electron microscopy. In: Current Protocols in Cell Biology, Bonifacino, J. S. Dasso, M. Harford, J. B. Lippincott-Schwartz, J. and Yamada, K. M. (Eds.), John Wiley & Sons, New York, 4.8.1-4.8.9 (2001).
Procedure for thin sections and cryosections:
Bonfanti, L.; Mironov, A. A, Jr.; Martinez-Menarguez, J. A.; Martella, O.; Fusella, A.; Baldassarre, M.; Buccione, R.; Geuze, H. J.; Mironov, A. A., and Luini, A.: Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation. Cell, 95, 993-1003 (1998).
More information:
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Visintin and co-workers used Nanogold® labeling with silver enhancement in SEM studies on the function of the mammalian signaling receptor for bacterial lipopolysaccharide (LPS). Three cell-surface proteins have been recognized as components of this receptor: CD14, Toll-like receptor-4 (TLR4), and MD-2. MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes. Consequently, TLR4 clusters into receptosomes (many of which are massive) that recruit intracellular toll/IL-1/resistance domain-containing adapter proteins within minutes, thus initiating signal transduction; this process was visualized highly effectively by SEM after labeling with anti-TLR4 monoclonal antibody HTA125 bound by Nanogold-Fab' with silver enhancement. MD-2 must be bound to TLR4 on the cell surface before binding can occur; TLR4 activation correlates with the ability of MD-2 to bind LPS, as MD-2 mutants that still bind TLR4, but are impaired in the ability to bind LPS, conferred a greatly blunted LPS response. These findings help clarify the earliest events of TLR4 triggering by LPS and make MD-2 an attractive target for pharmacological intervention in endotoxin-mediated diseases.
Cells were grown on poly-L-lysine-treated coverslips and treated with 1 microgram/mL LPS for 5 min at 37°C. Samples were chilled on ice, washed with ice-cold Hanks balanced saline solution, fixed for 20 minutes in 4% glutaraldehyde, then stained for 30 min with monoclonal antibody HTA125 (1 microgram/sample in 200 microliters). Antigen-antibody complexes were then revealed by a 30-minute incubation with Nanogold anti-mouse Fab' polyclonal antibody followed by enhancement with LI Silver. The silver-enhanced coverslips were then washed twice for 5 min with ultrapure water, dehydrated through a graded series of ethanol soaks to 100%, and then critical point-dried in liquid carbon dioxide. The coverslips with the dried cells were mounted and gold-coated for scanning electron microscopy, and examined on an Etec Autoscan electron microscope at 20 kV. Atomic contrast imaging was performed on the samples backscatter to confirm the silver nature of the white particles.
Reference:
Visintin, A.; Latz, E.; Monks, BG.; Espevik, T., and Golenbock, D. T.: Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction. J. Biol. Chem., 278, 48313-48320 (2003).
More information:
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The other advantage of Nanogold is that it is conjugated by chemical cross-linking rather than absorption, and this enables their conjugation to specific sites within biological molecules where their properties impart useful functionality. This is potentially very important for DNA, since the mechanism of hybridization enables the programmed assembly of complex structures. For example, Hamad-Schifferli and co-workers used amino-modified molecular beacons labeled with Mono-Sulfo-NHS Nanogold to achieve remote control of DNA hybridization, by means of the highly localized temperature rise produced by the conjugated Nanogold® particle inductively coupled to a pulsed radio frequency magnetic field. The effect was fully reversible and highly localized.
Reference:
Hamad-Schifferli, K.; Schwartz, J. J.; Santos, A. T.; Zhang, S., and Jacobson, J. M.: Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna. Nature, 415, 152-155 (2002).
Kiehl and co-workers have described the self-assembly of metallic nanoparticle arrays using DNA crystals, labeled site-specifically with Nanogold®, as a programmable molecular scaffolding; this represents a critical step toward the realization of DNA nanoelectronic applications. DNA-Nanogold conjugates were prepared from trityl-protected 5'-thiol-modified C6 oligonucleotides, which were deprotected and labeled with Monomaleimido Nanogold. DNA : Nanogold labeling stoichiometry of the purified conjugate was estimated spectroscopically to be very close to the desired 1:1 product.
Reference:
Xiao, S.; Liu, F.; Rosen, A. E.; Hainfeld, J. F.; Seeman, N. C.; Musier-Forsyth, K., and Kiehl, R. A.: Selfassembly of metallic nanoparticle arrays by DNA scaffolding. J. Nanoparticle Res., 4, 313-17 (2002).
For those who want to stick DNA onto conventional colloidal gold, Aqua and co-workers have investigated the interactions between DNA and gold particles; they found that pre-treatment with a displaceable layer of alkanethiols increases the selectivity with which thiolated oligonucleotides absorb to colloidal gold particles, and cuts down on other interactions.
Reference:
Aqua, T.; Naaman, R., and Daube, S. S.: Controlling the Adsorption and Reactivity of DNA on Gold. Langmuir, 19, 10573-10580 (2003).
Colloidal gold is almost always prepared by sodium citrate reduction, yet the role of citrate in stabilizing the gold and controlling the binding of biological molecules is not widely known. Lin and co-workers have conducted a combined scanning probe microscopy study of citrate adsorbed on the Au(111) surface, and imaged the adlayer structure of citrate on Au(111) using in situ scanning tunneling microscopy for the first time. Citrate anions form stable and well-ordered adlayers with (4 x 2 {SqRt3}) symmetry. The effect of the citrate adlayer on the interaction between the gold surface and fibrinogen, a protein with a major role in blood coagulation, was investigated by atomic force microscopy. Tightly bound fibrinogen on citrate-modified Au(111) features well-separated individual molecules, while fibrinogen adsorbed on bare Au(111) formed large aggregates of several molecules. These results indicate that citrate modification may change the biocompatibility of gold.
Reference:
Lin, Y.; Pan, G.-B. ; Su, G.-J. ; Fang, X.-H.; Wan, L.-J., and Bai, C.-L.: Study of Citrate Adsorbed on the Au(111) Surface by Scanning Probe Microscopy. Langmuir, 19, 10000-10003 (2003).
More information and help:
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Olofsson and co-workers describe a sensitive and easily regenerated nano-optical biosensor based on immobilization of avidin-coated 13.5 nm colloidal gold particles on a biotin-modified planar lipid bilayer supported on the walls of a quartz cuvette. This sensing template is specific for capturing of biotinylated biomacromolecules: analysis of changes in the optical spectrum upon binding of a biotinylated target, combined with Mie theory calculations, was used to quantitate the colorimetric changes induced by biorecognition events in the interfacial region of the particles. Agreement between theory and experiment was demonstrated using Feiters formalism, which correlates changes in effective refractive index and thickness with adsorbed mass. This template was sensitive enough to follow the hybridization kinetics of 15-mer fully complementary DNA strands, without the introduction of labels or secondary signal amplification.
Reference:
Olofsson, L.; Rindzevicius, T. ; Pfeiffer, I.; Käll, M., and Höök, F.: Surface-Based Gold-Nanoparticle Sensor for Specific and Quantitative DNA Hybridization Detection. Langmuir, 19, 10414-10419 (2003).
At Nanoprobes, we are working to bring some of the advantages of Nanogold® - site-specific linking, conjugation to novel probes, preparation of chemically selective labeling reagents - to larger gold probes. See some preliminary results using a covalently linked 10 nm gold-Fab' conjugate in our abstract from Microscopy and Microanalysis 1999. In the meantime, although we no longer make conventional colloidal gold, our technical help page is still available. Some other helpful resources are shown below.
More information:
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More new results from our collaborators at the Cleveland Clinic on in situ hybridization detection methods and reagents will be presented at the San Antonio Breast Cancer Symposium. Look for our presentation on "Quantitative image analysis of GOLDFISH, (a first generation gold-facilitated autometallographic bright field in-situ hybridization assay) for HER2 gene amplification in invasive breast cancer" on Saturday December 6 (poster #611).
More information:
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Here's a concept for fluorescent labeling - Fluorobodies, which combine the binding regions of antibodies with fluorescent proteins. Zeytun and co-workers describe them in the current issue of Nature Biotechnology: by inserting antibody binding loops into four of the exposed loops at one end of green fluorescent protein (GFP), they prepare small fluorescent probes with the binding characteristics of antibodies.
Reference:
Zeytun, A.; Jeromin, A.; Scalettar, B. A.; Waldo, G. S.; Bradbury, A. R.; Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies. Nat. Biotechnol., 21, 1473-1479 (2003).
Fudouzi and Xia describe the preparation of colloidal crystals with tunable colors, and their use as photonic papers. The voids within an opaline lattice of polystyrene beads were infiltrated with a liquid prepolymer to poly (dimethylsiloxane), then thermally cured. When a liquid capable of swelling the elastomer matrix was applied to the surface of this crystal, the lattice constant and thus the wavelength of Bragg-diffracted light was increased. In one example, the color of light diffracted from a colloidal crystal made of 175 nm polystyrene beads could be varied from violet to green, orange, and red simply by swelling with different solvents (silicone fluid, hexane, or octane). Color patterns could be conveniently generated on the surface of a thin film of colloidal crystal by writing with a Pilot pen, by screen printing, or by microcontact printing with an elastomer stamp.
Reference:
Fudouzi, H. and Xia, Y.: Colloidal Crystals with Tunable Colors and Their Use as Photonic Papers. Langmuir, 19, 9653-9660 (2003).
Menétrey and Dubayle report a novel one-step dual staining method for Mast cells, based on the differential metachromatic properties of proteoglycans, mostly heparin and chondroitin sulfate, and 1-naphthol in the presence of toluidine blue in an acidic medium. 1-naphthol is used to demonstrate the peroxidase activity of the sections treated by the horseradish peroxidase-labeled avidinbiotin complex method for antigen detection. Granules containing proteoglycans present the classical metachromatic reaction by appearing purplish-red, while granules containing antigen appear a brilliant green. When both types of granules are distinct inside the cell, single- and double-stained cells can be accurately separated and counted.
Reference:
Menétrey, D., and Dubayle, D.: A one-step dual-labeling method for antigen detection in mast cells. Histochem. Cell. Biol., 120, 435-442 (2003).
- Abstract (courtesy of Histochemistry and Cell Biology):
LINK
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