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Cytoplasmic Surface (cytoplasmic + surface)
Selected AbstractsExpression and distribution of ZO-3, a tight junction MAGUK protein, in mouse tissuesGENES TO CELLS, Issue 11 2003Akihito Inoko Background:, Three related MAGUK proteins, ZO-1, ZO-2 and ZO-3, are concentrated at the cytoplasmic surface of tight junctions. However, in contrast to ZO-1/ZO-2, our knowledge of the expression and distribution of ZO-3 is still fragmentary, partly due to a lack of antibodies that specifically distinguish ZO-3 from ZO-1 and ZO-2. Results:, We generated one pAb and one mAb that specifically recognized ZO-3 on Western blotting. The immunofluorescence signals obtained with these antibodies completely disappeared from ZO-1/ZO-2-positive tight junctions in the liver of ZO-3-deficient mice, indicating that the antibodies can be used to localize ZO-3 in various tissues by immunofluorescence microscopy. Immunofluorescence microscopy with these antibodies revealed that ZO-3 was concentrated at tight junctions in various types of epithelium, but not in endothelium or at cadherin-based cell-cell adhesion sites (spot adherens junctions of fibroblasts and intercalated discs of cardiac muscle cells), where ZO-1 and ZO-2 are concentrated. Conclusions:, We conclude that ZO-3 is expressed in a more epithelium-specific manner than ZO-1 and ZO-2. These observations provide for a better understanding of the functions of tight junction-associated MAGUKs. [source] pH-dependent translocation of ,-tocopherol transfer protein (,-TTP) between hepatic cytosol and late endosomesGENES TO CELLS, Issue 10 2003Masakuni Horiguchi Background:, ,-Tocopherol transfer protein (,-TTP), a member of the Sec14 protein family, plays an important role in transporting ,-tocopherol, a major lipid-soluble anti-oxidant, in the cytosolic compartment of hepatocytes and is known as a product of the causative gene for familial isolated vitamin E deficiency. It has been shown that the secretion of hepatocyte ,-tocopherol taken up with plasma lipoproteins is facilitated by ,-TTP. To explore the mechanism of ,-TTP mediated ,-tocopherol secretion, we investigated drugs which may affect this secretion. Results:, We found that, in a hepatocyte cell culture system, intracellular ,-tocopherol transport is impaired by chloroquine, an agent known for its function of elevating the pH in acidic compartments. Under chloroquine treatment, the diffuse cytosolic distribution of ,-TTP changes to a punctate pattern. Double-staining experiments with endocytosis markers revealed that ,-TTP accumulates transiently on the cytoplasmic surface of late endosomal membranes. This phenomenon is specific for hepatoma cell lines or primarily cultured hepatocytes. Other members of the Sec14 family, such as cellular retinaldehyde-binding protein (CRALBP) and supernatant protein factor (SPF), do not show this accumulation. Furthermore, we elucidate that the obligatory amino acid sequence for this function is located between amino acids 21 and 50, upstream of the N-terminal end of the lipid-binding domain. Conclusion:, We hypothesize that a liver-specific target molecule for ,-TTP exists on the late endosomal membrane surface. This transient binding may explain the mechanism of how ,-tocopherol is transferred from late endosomes to cytosolic ,-TTP. [source] Localization of a flavonoid biosynthetic polyphenol oxidase in vacuolesTHE PLANT JOURNAL, Issue 2 2006Eiichiro Ono Summary Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+ -translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower. [source] Subtle myelin defects in PLP-null mice ,GLIA, Issue 3 2006Jack Rosenbluth Abstract This study explores subtle defects in the myelin of proteolipid protein (PLP)-null mice that could potentially underlie the functional losses and axon damage known to occur in this mutant and in myelin diseases including multiple sclerosis. We have compared PLP-null central nervous system (CNS) myelin with normal myelin using ultrastructural methods designed to emphasize fine differences. In the PLP-null CNS, axons large enough to be myelinated often lack myelin entirely or are surrounded by abnormally thin sheaths. Short stretches of cytoplasm persist in many myelin lamellae. Most strikingly, compaction is incomplete in this mutant as shown by the widespread presence of patent interlamellar spaces of variable width that can be labeled with ferricyanide, acting as an aqueous extracellular tracer. In thinly myelinated fibers, interlamellar spaces are filled across the full width of the sheaths. In thick myelin sheaths, they appear filled irregularly but diffusely. These patent spaces constitute a spiral pathway through which ions and other extracellular agents may penetrate gradually, possibly contributing to the axon damage known to occur in this mutant, especially in thinly myelinated fibers, where the spiral path length is shortest and most consistently labeled. We show also that the "radial component" of myelin is distorted in the mutant ("diagonal component"), extending across the sheaths at 45° instead of 90°. These observations indicate a direct or indirect role for PLP in maintaining myelin compaction along the external surfaces of the lamellae and to a limited extent, along the cytoplasmic surfaces as well and also in maintaining the normal alignment of the radial component. © 2006 Wiley-Liss, Inc. [source] | ||||||||||