Putative silicon transport vesicles in the cytoplasm of the diatom Synedra acus during surge uptake of silicon

We studied the growth of the araphid pennate diatom Synedra acus subsp. radians (Kützing) Skabichevskii using a fluorescent dye N ¹,N ³-dimethyl-N ¹-(7-nitro-2,1,3-benzoxadiazol-4-yl)propane-1,3-diamine (NBD-N2), which stains growing siliceous frustules but does not stain other subcellular organelles. We used a clonal culture of S. acus that was synchronized by silicon starvation. Epifluorescence microscopy was performed in two different ways with cells stained by the addition of silicic acid and the dye. Individual cells immobilized on glass were observed during the first 15–20 min following the replenishment of silicic acid after silicon starvation. Alternatively, we examined cells of a batch culture at time intervals during 36 h after the replenishment of silicic acid using fluorescence and confocal microscopy. The addition of silicic acid and NBD-N2 resulted in the rapid (1–2 min) formation of several dozen green fluorescent submicrometer particles (GFSPs) in the cytoplasm, which was accompanied by the accumulation of fluorescent silica inside silica deposition vesicles (SDVs) along their full length. In 5–15 min, GFSPs disappeared from the cytoplasm. Mature siliceous valves were formed within the SDVs during the subsequent 14–16 h. In the next 8–10 h, GFSPs appeared again in the cytoplasm of daughter cells. The data obtained confirm observations about the two-stage mechanism of silicon assimilation, which includes rapid silicon uptake (surge uptake) followed by slow silica deposition. It is likely that the observed GFSPs are silicon transport vesicles, which were first proposed by Schmid and Schulz in (Protoplasma 100:267–288, 1979).     

Taxonomic characterization of the microorganisms associated with the cultivable diatom Synedra acus from Lake Baikal

Determination of the taxonomic position of microorganisms associated with diatoms was carried out using microscopic, microbiological, and phylogenetic methods. Examination of the cultures of the fresh-water diatom Synedra acus grown in Lake Baikal water by epifluorescence and electron microscopy revealed that bacterial cells colonized the mucilage surrounding the cells of living microalgae and their cell surfaces and penetrate into the frustules of dead diatoms. A total of 13 strains of heterotrophic bacteria were isolated in pure cultures and described. Based on the results of analysis of their morphological, physiological, and biochemical properties, as well as on data obtained by 16S rRNA gene analysis, the following strains were identified to the species level: Sphingomonas sp., Variovorax paradoxus, Pseudomonas fluorescens, Microbacterium trichothecenolyticum, Rhodococcus sp., Caulobacter vibrioides, and Brevundimonas vesicularis.     

Conservative motif CMLD in silicic acid transport proteins of diatom algae

Sequencing of fragments of genes coding for silicic acid transport (SIT) proteins of diatoms of evolutionary distant classes (centric Chaetoceros muelleri Lemmermann, pennate araphid Synedra acus Kützing, pennate raphid Phaeodactylum tricornutum Bohlin, and pennate with keeled raphe system Cylindrotheca fusiformis Reimann et Lewin), revealed the presence in these proteins of a conservative amino acid motif CMLD. Hydropathy profiles suggest that CMLD occupies a position between two transmembrane strands which do not contain lysine and arginine residues. The two strands are good candidates for the role of the channel along which transport of silicic acid occurs. CMLD is a rare motif. Diatoms are known to need Zn2+ for the incorporation of silica. Presumably, CMLD is the site of Zn2+ binding of SITs. We found that the growth of diatoms is inhibited by a negatively charged alkylating reagent 5-(2-iodoacetamidoethyl)aminonaphtalene-1-sulfonic acid which cannot penetrate through the cell membrane. Cysteine of CMLD can be a target of this reagent. Synthetic peptide NCMLDY forms a complex with Zn2+, as revealed by the fact that the ion considerably reduces the rate of alkylation of the peptide.     

Dissection of the frustules of the diatom Synedra acus under the action of picosecond impulses of submillimeter laser irradiation

Diatom algae realize highly intriguing processes of biosynthesis of siliceous structures in living cells under moderate conditions. Investigation of diatom physiology is complicated by frustule (siliceous exoskeleton). Frustules consist of valves and girdle bands which are adhered to each other by means of organic substances. Removal of the frustule from the lipid membrane of diatom cells would open new possibilities for study of silicon metabolism in diatoms. We found that submillimeter laser irradiation produced by a free-electron laser causes splitting of diatom frustules without destruction of cell content. This finding opens the way to direct study of diatom cell membrane and to isolation of cell organelles, including silica deposition vesicles. We suppose that the dissection action of the submillimeter irradiation results from unusual ultrasonic waves produced by the short (30–100 ps) but high-power (1 MW) terahertz laser impulses at 5.6 MHz frequency.     

Detection of KIR6 family protein in rat heart and liver mitochondria by immunoelectron microscopy

The electron microscopic study of thin sections of rat liver and heart using commercial specific antibodies against KIR.6.2 and secondary antibodies conjugated with colloidal gold was performed. It was found that the gold-labeled protein is localized in mitochondria of cardiomyocytes and hepatocytes but not in rough and smooth endoplasmic reticulum of hepatic cells and myofibrils of myocardium. In rat heart and liver mitochondria, the gold label was mainly located in mitochondrial cristae and was not found in mitochondrial matrix and intermembrane space. The data indicate that in heart and liver mitochondria there exists a protein similar in structure to the channel-forming subunit of a cytoplasmic potassium channel, KIR6.2. This is also supported by the presence of common modulators of cytoplasmic and mitochondrial ATP-dependent potassium channels. A possible role of the protein as a subunit of the mitochondrial ATP-dependent potassium channel is discussed.     

Selective induction of peroxisomal enzymes by the hypolipidemic drug bezafibrate. Detection of modulations by automatic image analysis in conjunction with immunoelectron microscopy and immunoblotting

Quantitative immunoelectron microscopy in conjunction with quantitative analysis of immunoblots have been used to study the effects of bezafibrate (BF), a peroxisome-proliferating hypolipidemic drug, upon six different enzyme proteins in rat liver peroxisomes (Po). Antibodies against following peroxisomal enzymes: catalase, urate oxidase, alpha-hydroxy acid oxidase, acyl-CoA oxidase, bifunctional enzyme (hydratase-dehydrogenase) and thiolase, were raised in rabbits, and their monospecificities were confirmed by immunoblotting. Female Sprague-Dawley rats were treated for 7 days with 250 mg/kg/day bezafibrate and liver sections were incubated with the appropriate antibodies followed by the protein A-gold complex. The labeling density for each enzyme was estimated by automatic image analysis. In parallel experiments immunoblots prepared from highly purified peroxisome fractions of normal and BF-treated rats were incubated with the same antibodies. The antigens were visualized by an improved protein A-gold method including an anti-protein A step and silver amplification. The immunoblots were also quantitated by an image analyzer. The results revealed a selective induction of beta-oxidation enzymes by bezafibrate with thiolase showing the most increase followed by bifunctional protein and acyl-CoA oxidase. The labeling density for catalase and alpha-hydroxy acid oxidase was reduced, confirming fully the quantitative analysis of immunoblots which in addition revealed reduction of uricase. These observations demonstrate that hypolipidemic drugs induce selectively the beta-oxidation enzymes while other peroxisomal enzymes are reduced. The quantitative immunoelectron microscopy with automatic image analysis provides a versatile, highly sensitive and efficient method for rapid detection of modulations of individual proteins in peroxisomes.     

Localization of the phosphocholine-binding sites on C-reactive protein by immunoelectron microscopy

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.     

Subcellular localization of DNA-binding protein BA by immunofluorescence and immunoelectron microscopy

Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined.     

Detection of desmin-containing intermediate filaments in cultured muscle and nonmuscle cells by immunoelectron microscopy

Antibodies raised against chicken gizzard smooth muscle desmin were shown to be specific by immunofluorescence cytochemistry and immunoautoradiography after two-dimensional polyacrylamide gel electrophoresis. Embryonic chick heart cell cultures (permeabilized with Triton X-100) and enucleated adult chicken erythrocyte ghosts (Granger, B. L., E. A. Rapasky, and E. Lazarides, 1982, J. Cell Biol. 92:299-312) were then used for immunoelectronmicroscopic localization of desmin. As expected, all intermediate filaments (IF) of the cardiac myocytes were labeled heavily and uniformly with the desmin antibodies. No periodicity or helicity was detectable along the labeled IF. Of interest was the intermittent but clear labeling of the IF of the nonmuscle, fibroblastic cells in the identical cultures. These antibodies did not bind vimentin from embryonic chick heart homogenates; furthermore, they did not label IF of avian erythrocytes known to contain vimentin but not desmin. We conclude that IF of cardiac fibroblastic cells contain low, but significant, concentrations of desmin and that this protein probably forms a copolymer with vimentin in these cells.     

Conserved motif CMLD in silicic acid transport proteins of diatoms

Sequenced fragments of genes coding for silicon transporters (SITs) were analyzed for diatoms of evolutionarily distant classes (centric Chaetoceros muelleri Lemmermann, pennate araphid Synedra acus Kützing, pennate raphid Phaeodactylum tricornutum Bohlin, and pennate Cylindrotheca fusiformis Reimann et Lewin with a keeled raphe system). SITs were found to contain a conserved motif, CMLD. Hydropathy profiles showed that the motif CMLD is between two transmembrane domains lacking Lys and Arg, and the domains were consequently assumed to play a role in the formation of a channel mediating silicic acid transport. The motif CMLD proved to be rare. Since Zn²⁺ is necessary for silica incorporation into diatom cells, a hypothesis was advanced that the motif CMLD acts as a Zn-binding site. Diatom growth suppression was observed in the presence of the alkylating agent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonic acid (AEDANS), which does not penetrate into the cell. Cys of the motif CMLD was assumed to act as a target for AEDANS. Zinc ions inhibited Cys alkylation in the synthetic peptide NCMLDY, testifying to the above hypothesis.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 303–316.Original Russian Text Copyright © 2005 by Sherbakova, Masyukova, Safonova, Petrova, Vereshagin, Minaeva, Adelshin, Triboy, Stonik, Aizdaitcher, Kozlov, Likhoshway, Grachev.     

Intramolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing

Three epitopes have been localized by immunoelectron microscopy on subunit Aa6 of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6:187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.     

Detection of intranuclear clusters of Sm antigens with monoclonal anti-Sm antibodies by immunoelectron microscopy

It has been shown that small nuclear RNA (snRNA) species U1, U2, U4, U5, and U6 are found in the nucleus in the form of small nuclear ribonucleoprotein particles (snRNPs), and that anti-Sm antibodies react with snRNP polypeptides, which are associated with all five snRNAs. We report here a novel intranuclear complex, denoted “Sm cluster,” detected by immunostaining with monoclonal anti-Sm antibodies in HeLa cells.     

Localisation of the protein and glycoprotein components of bovine nasal epithelial desmosomes by immunoelectron microscopy.

Desmosomal proteins (dp1-4) and glycoproteins (dg1-3) have been localised within desmosomes of bovine nasal epithelium by immunogold labelling of ultrathin frozen sections. Beginning in the extracellular space and proceeding through the plaque to the tonofilaments, the following localisations were found. Labelling for the 130,000 and 115,000 Mr glycoproteins (dg2 and dg3) was predominantly in the extracellular space, a location consistent with their proposed adhesive function. The glycoproteins of 175,000-164,000 Mr (dg1) were also found in the extracellular space and in addition had cytoplasmic domains extending throughout the cytoplasmic plaque. The 83,000 Mr protein (dp3) was located along the cytoplasmic face of the membrane and extended into the plaque, whereas an antibody which recognises both the 83,000 Mr protein (dp3) and the 75,000 Mr protein (dp4) gave labelling both in and beyond the plaque. Labelling for the high mol. wt proteins of Mr 250,000 and 215,000 (dp1 and dp2) was largely excluded from the plaque, and was located distally, adjacent to the tonofilaments. Hemidesmosomes could not be labelled with antibodies to dg1-3 or dp3 and 4, but some labelling was obtained with antibody to dp1 and 2.     

Alpha-fodrin in the adrenal gland: localization by immunoelectron microscopy

Localization of fodrin, the brain equivalent of spectrin (a protein constituent of the erythrocyte membrane cytoskeleton), was investigated at the ultrastructural level in rat adrenal gland. By use of an affinity purified antibody directed against the alpha-fodrin subunit, all chromaffin cells, cortical cells, nerve fibers, and their surrounding Schwann cells were found to be labeled close to the cytoplasmic side of their plasma membranes. The labeling appeared more intense for chromaffin cells, and secretory granules and mitochondria were frequently found to be associated with the zone containing alpha-fodrin in these cells. The immunostained zone was estimated to extend 230 +/- 70 nm into the cytoplasm. This localization is discussed in terms of what is known of the properties of spectrin, and possible roles of the molecule in the chromaffin cell are suggested.