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PubMed2000

Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+ channels.

The structure of the cytoplasmic assembly of voltage-dependent K+ channels was solved by x-ray crystallography at 2.1 angstrom resolution. The assembly includes the cytoplasmic (T1) domain of the integral membrane alpha subunit together with the oxidoreductase beta subunit in a fourfold symmetric T1(4)beta4 complex. An electrophysiological assay showed that this complex is oriented with four T1 domains facing the transmembrane pore and four beta subunits facing the cytoplasm. The transmembrane pore communicates with the cytoplasm through lateral, negatively charged openings above the T1(4)beta4 complex. The inactivation peptides of voltage-dependent K(+) channels reach their site of action by entering these openings.

Animals↗
PubMed2006

Atg9 sorting from mitochondria is impaired in early secretion and VFT-complex mutants in Saccharomyces cerevisiae.

In eukaryotic cells, the turnover of long-lived proteins and large cytoplasmic structures is mediated by autophagy. Components that have to be eliminated are sequestered into double-membrane vesicles called autophagosomes and delivered into the lysosome or vacuole where they are destroyed by resident hydrolases. The integral membrane protein Atg9 is essential for both autophagy and the cytoplasm-to-vacuole targeting pathway, a selective biosynthetic process in Saccharomyces cerevisiae that is mechanistically and morphologically similar to autophagy. Atg9 cycles between the pre-autophagosomal structure, the putative site of double-membrane vesicle biogenesis and mitochondria. To understand the function of Atg9, and also its trafficking mode between these two locations, we identified mutants that affect specific Atg9 transport steps. We recently reported that five Atg proteins and phosphatidylinositol-3-phosphate regulate Atg9 recycling from the pre-autophagosomal structure. Here, we describe a different category of mutants that blocks Atg9 sorting from mitochondria. All mutants have been previously shown to be required for the normal progression of both the Cvt pathway and autophagy, but their precise role in these transport routes was unknown.

Actins↗
PubMed1983

Dissipation of amino acid concentration gradients within rabbit ileal enterocytes.

The distribution of alanine, lysine and methionine within the cytoplasm of functionally mature enterocytes in rabbit ileum was measured by autoradiography after a short period of contact with tritiated substrate. Pronounced intracellular concentration gradients were noted for alanine and lysine, the concentration of these amino acids in the apical part of the enterocyte being 2- to 3-times that found near the base of the cell. No such concentration gradient was seen for methionine. Subsequent superfusion of the mucosal surface of the tissue with substrate-free medium caused intracellular concentration gradients for alanine and lysine to disappear. There was also a decrease in the enterocyte content of all three amino acids unassociated with backflux into the intestinal lumen. The ease with which intracellular concentration gradients for alanine and lysine can be manipulated is used as an argument against the possibility that their creation results from selective attachment to cytoplasmic structures in the apical part of the enterocyte.

Alanine↗
PubMed1978

Three-dimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin- and tubulin-containing structures.

Cytoskeletons prepared by Triton X-100 treatment of tissue culture cells appear in stereo electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. Microfilament bundles and also tonofilament-like bundles are readily discerned when present in the cell type. In addition thinner fibers, some of which branch (smallest diameter 30--40 A), as well as fibers of larger diameter, some of which correspond to microtubules, can be seen. Since such cytoskeletons are an open, membrane-free system, individual fibrous organizations can be identified by specific antibodies. An indirect immunoferritin procedure using antibodies to tubulin or actin visualizes microtubules or actin-containing structures. Stereo electron microscopy of cytoskeletons decorated with actin antibody reveals, in addition to the F-actin-containing microfilament bundles, an extended fine actin lattice. This actin net is displayed throughout the cytoplasm not only between the microfilament bundles but also in those regions of the cytoskeleton that in the intact cell correspond to the submembraneous regions. Thus all actin-containing fibrous cytoplasmic structures may be interconnected in the living cell.

Actins↗
PubMed2005

Claudin 7 expression and localization in the normal murine mammary gland and murine mammary tumors.

INTRODUCTION: Claudins, membrane-associated tetraspanin proteins, are normally associated with the tight junctions of epithelial cells where they confer a variety of permeability properties to the transepithelial barrier. One member of this family, claudin 7, has been shown to be expressed in the human mammary epithelium and some breast tumors. To set the stage for functional experiments on this molecule, we examined the developmental expression and localization of claudin 7 in the murine mammary epithelium and in a selection of murine mammary tumors. METHOD: We used real-time polymerase chain reaction, in situ mRNA localization, and immunohistochemistry (IHC) to examine the expression and localization of claudin 7. Frozen sections were examined by digital confocal microscopy for colocalization with the tight-junction protein ZO1. RESULTS: Claudin 7 was expressed constitutively in the mammary epithelium at all developmental stages, and the ratio of its mRNA to that of keratin 19 was nearly constant through development. By IHC, claudin 7 was located in the basolateral part of the cell where it seemed to be localized to discrete vesicles. Scant colocalization with the tight-junction scaffolding protein ZO1 was observed. Similar results were obtained from IHC of the airway epithelium and some renal tubules; however, claudin 7 did partly colocalize with ZO1 in EPH4 cells, a normal murine mammary cell line, and in the epididymis. The molecule was localized in the cytoplasm of MMTV-neu and the transplantable murine tumor cell lines TM4, TM10, and TM40A, in which its ratio to cytokeratin was higher than in the normal mammary epithelium. CONCLUSION: Claudin 7 is expressed constitutively in the mammary epithelium at approximately equal levels throughout development as well as in the murine tumors examined. Although it is capable of localizing to tight junctions, in the epithelia of mammary gland, airway, and kidney it is mostly or entirely confined to punctate cytoplasmic structures, often near the basolateral surfaces of the cells and possibly associated with basolateral membranes. These observations suggest that claudin 7 might be involved in vesicle trafficking to the basolateral membrane, possibly stabilizing cytoplasmic vesicles or participating in cell-matrix interactions.

Animals↗
PubMed1985

The intermediate filaments in human hepatocytes.

The intermediate filaments (IFs) in human hepatocytes were studied in biopsy specimens from patients with minimal histological changes. They were clearly visualized three-dimensionally by using polyethylene-glycol-embedding method after washing out cellular organelles and other components of cytoskeletons with a solution containing 0.15% Triton X-100 and saponin 0.5 mg/ml. We demonstrated that IFs were distributed throughout the cytoplasm in a meshwork fashion, attached to the junctional complex, encircled the bile canalicular lumen, and were directly attached to nucleus. Even after extensive washout of cytoplasmic structures, hepatocytes did not collapse, with the nuclei situated at due places in the cells. These findings may support the view that IFs play a role in nuclear positioning and in keeping the cytoplasmic space in human hepatocytes.

Adolescent↗
PubMed1992

Reevaluation of envelope profiles and cytoplasmic ultrastructure of mycobacteria processed by conventional embedding and freeze-substitution protocols.

The cell envelope architectures and cytoplasmic structures of Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-14C]acetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (approximately 25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures.

Cell Membrane↗
PubMed1984

Cysteamine depletes prolactin (PRL) but does not alter the structure of PRL-containing granules in the anterior pituitary.

Cysteamine causes a profound depletion of PRL in the anterior pituitary and in the systemic circulation, as measured by RIA and bioassay. However, electron microscopic study of PRL-containing cells in rat anterior pituitary does not reveal changes in secretory granule or cytoplasmic structure during the interval of depressed PRL content and of subsequent recovery to normal levels. In contrast to the results obtained by RIA, PRL-like immunoreactivity as detected by immunocyto-chemistry is present and similar to that of control preparations after cysteamine administration. We suggest that cysteamine alters PRL structure in secretory granules, probably by interacting with the disulfide bonds of PRL, thereby altering bioactivity and immunoreactivity. The presence of cysteamine-altered PRL in secretory granules does not seem to trigger degradation of granules by the lysosomal system.

Animals↗
PubMed1989

[Estrogen production in epithelial tumors of the ovary--identification of estrogen-synthesizing cells].

Morphological, fine structural and enzyme histochemical investigation of common epithelial tumors of the ovary was aimed at identifying the estrogen-synthesizing cells in the stroma of the tumor. Histological changes in cell condensation or "thecosis" and cell enlargement or "luteinization" were commonly observed in the stromal area of the tumor. Two types of stromal cells were found by electron microscopic study; one had characteristic organelles of steroid hormone-producing cells, and the other had a similar cytoplasmic structure to that of fibroblastic cells. Both types of cells had lipid droplets in the cytoplasm. The former seemed to be transformed from the latter. Steroidogenesis-related enzymes such as 3 beta-hydroxy steroid dehydrogenase (HSD) and 17 beta-HSD were demonstrated in the stromal cells. Electron microscopy revealed that these enzymes were localized at the intercrystal space and inner membrane of mitochondria with tubular crystal. These findings indicated that thecosis appeared in the stroma of the epithelial tumor, and the stromal cell acquired estrogen-producing potential.

Cytoplasm↗
PubMed1975

Electron microscopy of critical point dried whole cultured cells.

To determine the overall fine structure of whole, unsectioned cells, cells from rat embryos were cultured on Formvar, glutaraldehyde/osmium-fixed, transferred to grids, dehypdrated, critical point dried, then examined by transmission electron microscopy at either 80 or 1000 kV. In contrast to air-dried material, critical point dried cells revealed each component clearly and with excellent contrast. All normal cytoplasmic structures (including coated vesicles, polyribosomes, microtubules and other fine components) were readily identifiable. Extensive structures such as microtubules and the endoplasmic reticulum (which appear fragmented in sections) were well displayed. At 1000 kV the beam readily penetrated even the thick nuclear and perinuclear cell regions and produced exceptionally crisp images. The methods described provide a simplified approach to the study of overall cell fine structure.

Cell Nucleolus↗
PubMedDate not supplied

The natural subcellular surface structure of the bovine sperm cell.

We used atomic force microscopy (AFM), which utilizes a novel 3D image-contrast mechanism, to obtain nanometer-resolved, topographic data images of the natural surface structures of untreated bovine sperm cells. Freshly ejaculated, thawed, sonicated, and demembranated bovine sperm were adsorbed passively or by motility from suspension onto a coverglass substrate and directly imaged in normal air and saline environments without damaging the cells. Our AFM images of the surface structures of unfixed sperm imaged in normal air were consistent with previous electron microscope results on frozen or fixed sperm, demonstrating that the accurate preservation of small cellular structures is achievable using greatly simplified AFM sample preparation and imaging environments. Our AFM results also indicate that imaging sperm in physiologic buffer provides more native views of sperm due to the retention of cytoplasmic structures easily disrupted by drying forces. In addition, the AFM images show that numerous nanometersized subcellular structures of the sperm head and tail regions could be clearly visualized on rapidly prepared, unfixed, intact cells. Consequently, AFM should be considered a new tool for studying sperm structure abnormalities and monitoring the specific effects of, or damage caused by, various chemical reactants or other treatments on the structures of metabolically active or partially demembranated sperm. AFM is now emerging as an important new structural technique for imaging hydrated cells and organelles and, in addition, has the capabilities to physically "interrogate" them with the local probe.

Acrosome↗
PubMed1980

Binding sites of fluorescent derivatives of insulin in nuclei, rough endoplasmic reticulum, and mitochondria.

Intracellular insulin-binding sites were directly traced in fixed monolayer cultures of a variety of cell types with the use of two fluorescent derivatives of insulin, viz. fluorescein isothiocyanate (FITC)-labelled and tetramethyl rhodamine isothiocyanate (TMRITC)-labelled insulin. Both derivatives retained the property of stimulating DNA synthesis in fibroblasts. Insulin-binding sites were found in the nuclear envelope, nucleoplasm, nucleoli, and in mitochondria and rough endoplasmic reticulum. The identity of these structures was established by concomitant studies on the same cell by means of phase contrast optics and immunocytochemical tracing with specific antibodies to nuclei, mitochondria, or ribosomes. Binding of insulin to the nuclear and cytoplasmic structures was rapid, reversible and saturable, temperature and pH-dependent, and inhibited by an excess of native, but not other, hormones. The staining reactions were sensitive to treatment by the nonionic detergents, NP-40 and TX-100, and to trypsin and pronase, but not to DNase and RNase, suggesting that the binding sites are protein in nature.

Animals↗
PubMed1990

Subcellular distribution of Na+/H+ antiport activity in rat renal cortex.

Phase partitioning analyses of a brush border membrane preparation obtained with a divalent cation precipitation procedure (Am J Physiol 246:F853-F858, 1984) confirmed that Na+/H+ antiport activity was localized primarily to the brush border membrane of the rabbit proximal tubular epithelial cell. This analysis also indicated that antiport activity was associated with membrane populations that appeared to be derived from cytoplasmic structures. However, since the starting point of the analysis was a partially-purified brush border sample rather than a total membrane sample, it was not possible to discern the magnitude of the potential cytoplasmic pool of antiport activity. We have now used a three dimensional analytical fractionation procedure, based on differential centrifugation, equilibrium density gradient centrifugation, and partitioning in an aqueous polymer 2-phase system, to survey the subcellular distribution of Na/H antiport activity in rat kidney cortex. Roughly 53% of the recovered antiport activity could be assigned to a population of brush border membrane vesicles characterized by a 15-fold enrichment of maltase. An additional 26% of the recovered activity could be assigned to a group of three membrane populations whose biochemical characteristics appeared equally consistent with origins in distinct microdomains of the brush border membrane and with origins in microdomains of the Golgi complex involved in the assembly or recycling of brush border membrane constituents. Therefore, depending on the identities of membranes which contained the secondary pool of Na+/H+ antiport activity, no more than one-third of the total recoverable Na+/H+ antiport activity could be assigned to cytoplasmic membranes of the proximal tubular epithelium.

Animals↗
PubMed1978

Diagnostic electron microscopy of head and neck tumors.

One hundred sixty-one neoplasms from the region of the head and neck were studied by electron microscopy to define (1) subcellular structures important of essential to diagnosis, and (2) the application and/or limitations of fresh, formaldehyde-fixed, and paraffin-blocked tissues for diagnostic use. It was concluded that electron microscopical identification of specific cytoplasmic structures was helpful in the diagnosis of head and neck neoplasms, especially those that were equivocal at the light microscopical level. Although electron microscopy could permit a more accurate classification of neoplasms in many cases, it was of little value in separating benign from malignant lesions. It was also concluded that formaldehyde-fixed tissue was a reliable source of material for these studies. Paraffin-embedded tissue was much more limited.

Cytoskeleton↗
PubMed2004

Identification and characterization of evolutionarily conserved pufferfish, zebrafish, and frog orthologs of GASZ.

We previously identified Gasz (a germ cell-specific gene encoding a protein containing four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper) in six mammalian species. Here, we report GASZ orthologs in pufferfish (Fugu rubripes), zebrafish (Danio verio), and frog (Xenopus laevis). Sequences of the three Gasz cDNAs were determined by database mining and 5'- and 3'-rapid amplification of cDNA ends (RACE) followed by sequencing. The three orthologous vertebrate genes encode proteins structurally similar to mammalian GASZ and contain the characteristic four ankyrin repeats (ANKs) and sterile-alpha motif (SAM). Their ANK and SAM domains share 55- 74% and 38-55% amino acid identity with those in human GASZ, respectively. Similar to human and mouse Gasz genes, pufferfish Gasz is composed of 13 exons, spanning approximately 12 kilobases, and flanked by Cftr at its 5'-end and Wnt2 at its 3'-end. Northern and Western blot analyses detect frog Gasz expression only in testis and ovary. In situ hybridization and immunohistochemical analyses show that frog Gasz mRNA and protein expression is confined to pachytene spermatocytes in the testis and to oocytes in the ovary. In frog oocytes, GASZ protein appears to localize to a cytoplasmic structure resembling the Balbiani body, a postulated mRNA transport organizer in the cytoplasm. The high evolutionary conservation and germ cell specificity suggest that GASZ plays an essential role in gametogenesis. The data presented here are important for future studies of the physiological roles of GASZ using fish and amphibians as animal models.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells.

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.

Animals↗
PubMed2004

Cytoplasmic foci are sites of mRNA decay in human cells.

Understanding gene expression control requires defining the molecular and cellular basis of mRNA turnover. We have previously shown that the human decapping factors hDcp2 and hDcp1a are concentrated in specific cytoplasmic structures. Here, we show that hCcr4, hDcp1b, hLsm, and rck/p54 proteins related to 5'-3' mRNA decay also localize to these structures, whereas DcpS, which is involved in cap nucleotide catabolism, is nuclear. Functional analysis using fluorescence resonance energy transfer revealed that hDcp1a and hDcp2 interact in vivo in these structures that were shown to differ from the previously described stress granules. Our data indicate that these new structures are dynamic, as they disappear when mRNA breakdown is abolished by treatment with inhibitors. Accumulation of poly(A)(+) RNA in these structures, after RNAi-mediated inactivation of the Xrn1 exonuclease, demonstrates that they represent active mRNA decay sites. The occurrence of 5'-3' mRNA decay in specific subcellular locations in human cells suggests that the cytoplasm of eukaryotic cells may be more organized than previously anticipated.

Cell Compartmentation↗
PubMedDate not supplied

[Electron-autoradiographic and biochemical study of the role of foam cells in low density lipoprotein metabolism].

Participation of foam cells in metabolism of lipoproteins of low density (LPLD) was studied by biochemical methods and by electronic autoradiography. The foam cells were isolated from atherosclerotic rabbit aorta after perfusion with 12I-LPLD within 6 hrs. Metabolic activity of foam cells was evaluated by the ratio of radioactivity in lipid and protein components of cells as well as by distribution of reduced argentum granules among the subcellular structures on autoradiogram. The biochemical studies showed that the lipid components of LPLD were preferably accumulated in foam cells. Electronic-autoradiography demonstrated that protein from the lipoprotein particles was also distributed intracellularly in various cytoplasmic structures. Foam cells appear to capture intact lipoprotein particles by pinocytosis with subsequent splitting of protein component by cellular lysosome apparatus.

Animals↗