3-D analysis of bacterial cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches.

The formation of cell-(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2-D microscopy techniques, the 3-D and internal structure remain obscure. In this study, we examined the 3-D structure of cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3-D microscopy techniques. We obtained 3-D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4-200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike- or platelet-shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell-(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell-(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3-D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X-ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a comprehensive understanding of structure and composition of the various Fe-precipitates and their association with bacterial cells and EPS.

Regulated trafficking of cellulose synthases.

New findings reveal that proteins involved in cellulose biosynthesis undergo regulated trafficking between intracellular compartments and the plasma membrane. The coordinated secretion and internalization of these proteins involve both the actin and cortical microtubule cytoskeletons. This regulated trafficking allows the dynamic remodeling of cellulose synthase complex (CSC) secretion during cell expansion and differentiation. Several new actors of the cellulose synthesis machinery have been recently identified.

Chemical composition of the attachment pad secretion of the locust Locusta migratoria.

This study is the first attempt to characterise the chemical composition of the secretion of the smooth pads of the locust Locusta migratoria and to relate this to the composition of the cuticle coverage of the pads and the wings. Gas-chromatography and mass-spectrometry (GC-MS) were the principal techniques used for the characterization of these materials. Secretion droplets were visualised and quantified with the aid of diverse microscopic techniques. The chemical composition of prints is shown to differ from the cuticle coverage, in particular, with respect to the fatty acid distribution: in the secretion, saturated and unsaturated fatty acids with chain lengths between C(16) and C(20) in both the free form and as glycerides predominate, whereas cuticle coverage contains waxes of long-chained fatty-acids bound to long-chain primary alcohols. The second important difference is the significant amount of glucose and other saccharides found in methanolyzates of the pad fluid. A considerable amount of the amino acids (up to 53%) was detected in the non-volatile portion of the fluid. Data obtained from the shock-freezing, carbon-platinum coating and replica preparation show that the secretory droplets contain nano-droplets on their surfaces. The results lead us to suggest that the pad secretion is an emulsion consisting of lipidic nano-droplets dispersed in an aqueous liquid. According to the chemical composition of the secretion, a high-viscosity of the fluid may be suggested. Presumably, the fluid is a kind of a coupling agent, promoting and strengthening adhesion between otherwise incompatible materials by providing the proximity of contact for intermolecular forces.

Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.

Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.

Cell cycle-independent expression of the Arabidopsis cytokinesis-specific syntaxin KNOLLE results in mistargeting to the plasma membrane and is not sufficient for cytokinesis.

The Arabidopsis KNOLLE gene encodes a cytokinesis-specific syntaxin that localises to the plane of division and mediates cell-plate formation. KNOLLE mRNA and protein expression is tightly regulated during the cell cycle. To explore the significance of this regulation, we expressed KNOLLE protein under the control of two constitutive promoters, the flower-specific AP3 and the cauliflower mosaic virus 35S promoter. The transgenic plants developed normally, although KNOLLE mRNA and protein accumulated to high levels in non-proliferating cells and protein was incorporated into membranes. Immunolocalisation studies in transgenic seedling roots revealed mistargeting of KNOLLE protein to the plasma membrane in tip-growing root hairs and in expanding root cells, whereas no mislocalisation was observed in proliferating cells. By comparative in situ hybridisation to embryo sections, the 35S promoter yielded, relative to the endogenous KNOLLE promoter, low levels of KNOLLE mRNA accumulation in proliferating cells that were insufficient to rescue cytokinesis-defective knolle mutant embryos. Our results suggest that in wild type, strong expression of KNOLLE protein during M phase is necessary to ensure efficient vesicle fusion during cytokinesis.

Auxin transport inhibitors block PIN1 cycling and vesicle trafficking.

Polar transport of the phytohormone auxin mediates various processes in plant growth and development, such as apical dominance, tropisms, vascular patterning and axis formation. This view is based largely on the effects of polar auxin transport inhibitors. These compounds disrupt auxin efflux from the cell but their mode of action is unknown. It is thought that polar auxin flux is caused by the asymmetric distribution of efflux carriers acting at the plasma membrane. The polar localization of efflux carrier candidate PIN1 supports this model. Here we show that the seemingly static localization of PIN1 results from rapid actin-dependent cycling between the plasma membrane and endosomal compartments. Auxin transport inhibitors block PIN1 cycling and inhibit trafficking of membrane proteins that are unrelated to auxin transport. Our data suggest that PIN1 cycling is of central importance for auxin transport and that auxin transport inhibitors affect efflux by generally interfering with membrane-trafficking processes. In support of our conclusion, the vesicle-trafficking inhibitor brefeldin A mimics physiological effects of auxin transport inhibitors.

Characterization of a differentially expressed protein that shows an unusual localization to intracellular membranes in Leishmania major.

The SHERP genes are found as a tandem pair within the differentially regulated LmcDNA16 locus of Leishmania major. The SHERP gene product (small hydrophilic endoplasmic reticulum-associated protein) is unusual in its small size (6.2 kDa), its acidic pI (4.6) and its exclusive, high-level expression ( approximately 100000 copies per cell) in infective non-replicative parasite stages. No homologues have been found to date. Secondary-structure predictions suggest that SHERP contains an amphiphilic alpha-helix that is presumably involved in protein-protein interactions. SHERP has been localized to the endoplasmic reticulum as well as to the outer mitochondrial membrane in both wild-type and over-expressing parasites. Given the absence of an N-terminal signal sequence, transmembrane-spanning domains or detectable post-translational modifications, it is likely that this hydrophilic molecule is a peripheral membrane protein on the cytosolic face of intracellular membranes. This weak membrane association has been confirmed in cell-fractionation assays, in which SHERP redistributes from the cytoplasmic to the membrane fraction after in vivo cross-linking. SHERP does not appear to be involved in rearrangements of the cytoskeleton or conservation of organelle morphology during parasite differentiation. The role of this novel protein, presumed to be part of a protein complex, in infective parasites that are nutrient-deficient and pre-adapted for intracellular survival in the mammalian host is under investigation.

The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase.

The Leishmania ATP-binding cassette (ABC) transporter PGPA is involved in metal resistance (arsenicals and antimony), although the exact mechanism by which PGPA confers resistance to antimony, the first line drug against Leishmania, is unknown. The results of co-transfection experiments, transport assays, and the use of inhibitors suggest that PGPA recognizes metals conjugated to glutathione or trypanothione, a glutathione-spermidine conjugate present in Leishmania. The HA epitope tag of the influenza hemagglutinin as well as the green fluorescent protein were fused at the COOH terminus of PGPA. Immunofluorescence, confocal, and electron microscopy studies of the fully functional tagged molecules clearly indicated that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis and exocytosis in this parasite. Subcellular fractionation of Leishmania tarentolae PGPAHA transfectants was performed to further characterize this ABC transporter. The basal PGPA ATPase activity was determined to be 115 nmol/mg/min. Transport experiments using radioactive arsenite-glutathione conjugates clearly showed that PGPA recognizes and actively transports thiol-metal conjugates. Overall, the results are consistent with PGPA being an intracellular ABC transporter that confers arsenite and antimonite resistance by sequestration of the metal-thiol conjugates.

The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells.

The cellular distribution of two glycosyl-phosphatidylinositol (GPI)-anchored proteins and a trans-membrane protein and the compartments involved in their trafficking were investigated in the insect stage of Leishmania mexicana, which belongs to the phylogenetically old protozoan family Trypanosomatidae. Electron microscopy of sections from high-pressure frozen and freeze-substituted cells allowed a detailed description of exo- and endocytic structures located in the vesicle-rich, densely packed anterior part of the spindle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, the only site of endo- and exocytosis. A tubulovesicular compartment lined by one or two distinct microtubules and extending along the length of the cell is proposed to be a post-Golgi and probably late endosomal/lysosomal compartment. Using biotinylation experiments, FACS analysis and quantitative immunoelectron microscopy it was found that, at comparable expression levels, 73-75% of the two GPI-anchored proteins but only 13% of the trans-membrane protein are located on the cell surface. The tubulovesicular compartment contains 46%, the ER 5%, the Golgi complex 1.9% and the tubular cluster/translucent vesicle complex 3.6% of the intracellular fraction of the GPI-anchored protease, GP63. The density of GP63 was found to be 23-fold higher on the plasma/flagellar pocket membrane than on the ER and about tenfold higher than on membranes of the Golgi complex or of endo- or exocytic vesicles. These results indicate that there is a considerable concentration gradient of GPI-anchored proteins between the plasma/flagellar pocket membrane and the ER as well as structures involved in exo- or endocytosis. Possible mechanisms how this concentration gradient is established are discussed.

Isolation and characterization of glycosylphosphatidylinositol-anchored, mucin-like surface glycoproteins from bloodstream forms of the freshwater-fish parasite Trypanosoma carassii.

Wild and farmed freshwater fishes are widely and heavily parasitized by the haemoflagellate Trypanosoma carassii. In contrast, common carp, a natural host, can effectively control experimental infections by the production of specific anti-parasite antibodies. In this study we have identified and partially characterized mucin-like glycoproteins which are expressed in high abundance [(6. 0+/-1.7)x10(6) molecules.cell(-1)] at the surface of the bloodstream trypomastigote stage of the parasite. The polypeptide backbone of these glycoproteins is dominated by threonine, glycine, serine, alanine, valine and proline residues, and is modified at its C-terminus by a glycosylphosphatidylinositol membrane anchor. On average, each polypeptide carries carbohydrate chains composed of about 200 monosaccharide units (galactose, N-acetylglucosamine, xylose, sialic acid, fucose, mannose and arabinose), which are most probably O-linked to hydroxy amino acids. The mucin-like molecules are the target of the fish's humoral immune response, but do not undergo antigenic variation akin to that observed for the variant surface glycoprotein in salivarian trypanosomes. The results are discussed with reference to the differences between natural and experimental infections, and in relation to the recently delineated molecular phylogeny of trypanosomes.

Proteophosphoglycans of Leishmania mexicana. Identification, purification, structural and ultrastructural characterization of the secreted promastigote proteophosphoglycan pPPG2, a stage-specific glycoisoform of amastigote aPPG.

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans that appear to be important for successful colonization of the sandfly and for virulence in the mammalian host. A hallmark of these molecules is extensive phosphoglycosylation by phosphoglycan chains via the unusual linkage Manalpha1-PO(4)-Ser. In this study we have identified and purified to apparent homogeneity a novel proteophosphoglycan (pPPG2) which is secreted by Leishmania mexicana promastigotes (sandfly stage). Amino acid analysis and immunoblots using polypeptide-specific antisera suggest that pPPG2 shares a common protein backbone with a proteophosphoglycan (aPPG) secreted by Leishmania mexicana amastigotes (mammalian stage). Both pPPG2 and aPPG show a similar degree of Ser phosphoglycosylation (50. 5 mol% vs. 44.6 mol%), but the structure of their phosphoglycan chains is developmentally regulated: in contrast to aPPG which displays unique, complex and highly branched glycan chains [Ilg, Craik, Currie, Multhaup, and Bacic (1998) J. Biol. Chem. 273, 13509-13523], pPPG2 contains short unbranched structures consisting of >60 mol% neutral glycans, most likely (Manalpha1-2)(0-5)Man and Galbeta1-4Man, as well as about 40 mol% monophosphorylated glycans of the proposed structures PO(4)-6Galbeta1-4Man and PO(4)-6(Glcbeta1-3)Galbeta1-4Man. The major differences between pPPG2 and aPPG with respect to their apparent molecular mass, their ultrastructure and their proteinase sensitivity are most likely a consequence of this stage-specific glycosylation of their common protein backbone.

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors.

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.

Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.

Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions.

Protein kinases in control of the centrosome cycle.

The centrosome is the major microtubule nucleating center of the animal cell and forms the two poles of the mitotic spindle upon which chromosomes are segregated. During the cell division cycle, the centrosome undergoes a series of major structural and functional transitions that are essential for both interphase centrosome function and mitotic spindle formation. The localization of an increasing number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling many of these transitions. Here, we focus on two protein kinases, the polo-like kinase 1 and the NIMA-related kinase 2, for which recent data indicate key roles during the centrosome cycle.

Trypanosoma brucei: killing of bloodstream forms in vitro and in vivo by the cysteine proteinase inhibitor Z-phe-ala-CHN2.

Cysteine proteinases were tested for their suitability as targets for chemotherapy of sleeping sickness using the peptidyl inhibitor Z-Phe-Ala-diazomethyl ketone (Z-Phe-Ala-CHN2). In vitro, the inhibitory concentration of Z-Phe-Ala-CHN;2 required to reduce the growth rate by 50% was 400 times lower for culture-adapted bloodstream forms of Trypanosoma brucei than for a mouse myeloma cell line. At an inhibitor concentration of 10;M the parasites were lysed within 48 h of incubation. Parasitemia of mice infected with T. brucei decreased to undetectable levels for 3 days following treatment with 250 mg/kg Z-Phe-Ala-CHN2 on days 3 to 6 after infection. Although parasitemia returned thereafter to control levels, infected mice treated with the inhibitor survived approximately twice as long as those treated with placebo. Z-Phe-Ala-CHN2 inhibited proteinolysis in lysosomes in vitro and almost completely blocked cysteine proteinase activity in vivo. The results demonstrate the importance of cysteine proteinase activity for survival of T. brucei and suggest that such activity is an appropriate target for antitrypanosomal chemotherapy.

Isocitrate lyase of Ashbya gossypii--transcriptional regulation and peroxisomal localization.

The isocitrate lyase-encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C-terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C2 compounds ethanol and acetate, and fully induced by soybean oil.

Structure of a filamentous phosphoglycoprotein polymer: the secreted acid phosphatase of Leishmania mexicana.

The insect stage of the protozoan parasite Leishmania mexicana secretes a filamentous acid phosphatase (secreted acid phosphatase, SAP), a polymeric phosphoglycoprotein. The wild-type (wt) SAP filament is a copolymer composed of two related gene products SAP1 and SAP2, which are identical in the enzymatically active NH2-terminal domain and the COOH-terminal domain, but differ in the length of a highly glycosylated Ser/Thr-rich repeat region (32 amino acids and 383 amino acids, respectively) which is located between these domains. When expressed separately, full length SAP1, SAP2, or the NH2-terminal domain alone, are able to assemble into filaments. The Ser/Thr-rich region is the exclusive target for a novel type of O-glycosylation via phosphoserines. By using glycerol spraying/low-angle rotary metal shadowing and labelling with monoclonal antibodies it is demonstrated that the repetitive region adopts an extended conformation forming side arms which project radially from the filament core and terminate with the COOH-terminal domain. The length of the side arms of SAP1 and SAP2 (20 nm and 90 nm, respectively) corresponds to the predicted length of the Ser/Thr-rich repeat region of SAP1 and SAP2. Mass determination by scanning electron microscopy (STEM) shows that one morphologically defined globular particle of the filament core is a polypeptide dimer. We propose a model for the filament core, in which the globular NH2-terminal SAP domains form one strand composed of polypeptide dimers or two tightly associated strands of monomers which may twist into a double helix, similar to actin filaments. The highly O-glycosylated side arms project from the filament core conferring an overall bottle-brush-like appearance. The L. mexicana SAP is compared to SAPs secreted by the closely related species L. amazonensis and L. donovani.

Action of gold chloride ("gold toning") on silver-enhanced 1 nm gold markers.

In conventional immunoelectron microscopy (IEM), very small colloidal gold particles (0.8-3 nm), or the gold compound Nanogold (1.4 nm) are silver-enhanced for easy detection. However, silver enhancement has drawbacks. First, the silver layer is dissolved during fixation with osmium tetroxide, even if the concentration and incubation time are strongly reduced during pre-embedding labeling experiments in transmission electron microscopic (TEM) and scanning electron microscopic (SEM) studies. Second, after exposure to the electron beam the silver layer may migrate on the section or the whole particles may disappear. Sometimes silver migration can be observed even without irradiation. This effect strongly hampers reinvestigation of previously inspected areas, after some time of storage. In both cases, gold chloride treatment after silver enhancement is sufficient to completely protect the silver-enhanced 1 nm gold markers. Gold chloride treatment is part of the so-called "gold toning" procedure, which is a method used to substitute and/or cover the silver by a layer of gold. It can be applied in TEM and SEM experiments. As a serious drawback, gold chloride treatment slightly reduces the size of both unenhanced and silver-enhanced gold particles and can lead to disintegrated silver/gold particles. Therefore, this technique is useful for pre-embedding IEM, on-(resin)section, and ultrathin cryosection labeling experiments. However, it appears to be unsuitable for double-labeling studies using different gold sizes, for quantitation experiments, and in SEM.