scholarly journals SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney collecting duct principal cells

2001 ◽  
Vol 281 (3) ◽  
pp. F546-F556 ◽  
Author(s):  
Alok Shukla ◽  
Henrik Hager ◽  
Thomas Juhl Corydon ◽  
Andrew J. Bean ◽  
Ronald Dahl ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Northern Blotting ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Rt Pcr

The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins ( N-ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To examine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combined with DNA sequence analysis and Northern blotting using a digoxigenin-labeled Hrs-2 RNA probe. RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in rat kidney was confirmed by immunoblotting, revealing a 115-kDa protein in kidney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC-PK1 cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainly localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localization of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal laser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron segments. The labeling was predominantly present in intracellular vesicles, but labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 trafficking.

scholarly journals Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement

2013 ◽  
Vol 94 (3) ◽  
pp. 682-686 ◽  
Author(s):  
Kazuya Ishikawa ◽  
Kensaku Maejima ◽  
Ken Komatsu ◽  
Osamu Netsu ◽  
Takuya Keima ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Movement Protein ◽  
Rna Virus ◽  
Cell Movement ◽  
Plant Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Series Of Experiments ◽  
Fig Mosaic Virus

Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

Localization and regulation of PKA-phosphorylated AQP2 in response to V2-receptor agonist/antagonist treatment

2000 ◽  
Vol 278 (1) ◽  
pp. F29-F42 ◽  
Author(s):  
Birgitte Mønster Christensen ◽  
Marina Zelenina ◽  
Anita Aperia ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Fold Increase ◽  
Apical Plasma Membrane ◽  
Complete Disappearance ◽  
Vasopressin Secretion ◽  
V2 Receptor ◽  
Intracellular Vesicles

Phosphorylation of Ser256, in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser256 were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys1,d-Arg8]vasopressin (DDAVP) treatment or V2-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V2-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser256 is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V2 receptors by altering phosphorylation and/or dephosphorylation of Ser256in AQP2.

Immunocytochemical localization of Na+ channels in rat kidney medulla

1989 ◽  
Vol 256 (2) ◽  
pp. F366-F369 ◽  
Author(s):  
D. Brown ◽  
E. J. Sorscher ◽  
D. A. Ausiello ◽  
D. J. Benos
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Na Channels ◽  
Kidney Medulla ◽  
Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization

2004 ◽  
Vol 286 (3) ◽  
pp. F564-F571 ◽  
Author(s):  
Carsten Kneuer ◽  
Kerstin U. Honscha ◽  
Walther Honscha
Keyword(s):  
Laser Scanning ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Functional Characterization ◽  
Regulatory Elements ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Strong Inhibitor ◽  
Major Band ◽  
Sodium Dependent

Previous Northern blot studies suggested strong expression of a homolog to the sodium-dependent hepatocellular methotrexate transporter in the kidneys. Here, we report on the cloning of the cDNA for the renal methotrexate carrier isoform-1 (RK-MTX-1) and its functional characterization. Sequencing revealed 97% homology to the rat liver methotrexate carrier with an identical open reading frame. Differences were located in the 5′-untranslated region and resulted in the absence of putative regulatory elements (Barbie box, Ah/ARNT receptor) identified in the cDNA for the hepatocellular carrier. For functional characterization, MTX-1 cDNA was stably expressed in Madin-Darby canine kidney (MDCK) cells. A sodium-dependent transport of methotrexate with a Kmof 41 μM and a Vmaxof 337 pmol·mg protein-1·min-1was observed. This uptake was blocked by the reduced folates dihydro- and tetrahydrofolate as well as by methotrexate itself. Folate was inhibiting only weakly, whereas 5-methyltetrahydrofolate was a strong inhibitor. Further inhibitors of the methotrexate transport included the bile acids cholate and taurocholate and xenobiotics like bumetanide and BSP. PAH, ouabain, bumetanide, cholate, taurocholate, and acetyl salicylic acid were tested as potential substrates. However, none of these substances was transported by MTX-1. Furthermore, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells fivefold. Analysis of a fusion protein of RK-MTX-1 and the influenza virus hemagglutinin epitope by immunoblotting revealed a major band at 72 kDa within the cell membrane but not in the soluble fraction of transfected MDCK. Indirect immunofluorescence staining revealed an exclusive localization of the carrier in the plasma membrane, and by confocal laser-scanning microscopy we were able to demonstrate that the protein is expressed in the serosal region of MDCK tubules grown in a morphogenic collagen gel model.

scholarly journals Stay in Touch—The Cortical ER of Moss Protonemata in Osmotic Stress Situations

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 421 ◽  
Author(s):  
Dominik Harant ◽  
Ingeborg Lang
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Laser Scanning ◽  
Structural Changes ◽  
Physcomitrella Patens ◽  
Dynamic Network ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Natural State ◽  
Cell Cortex

Plasmolysis is usually introduced to cell biology students as a tool to illustrate the plasma membrane: hypertonic solutions cause the living protoplast to shrink by osmotic water loss; hence, it detaches from the surrounding cell wall. What happens, however, with the subcellular structures in the cell cortex during this process of turgor loss? Here, we investigated the cortical endoplasmic reticulum (ER) in moss protonema cells of Physcomitrella patens in a cell line carrying a transgenic ER marker (GFP-HDEL). The plasma membrane was labelled simultaneously with the fluorescent dye FM4-64 to achieve structural separation. By placing the protonemata in a hypertonic mannitol solution (0.8 M), we were able to follow the behaviour of the cortical ER and the protoplast during plasmolysis by confocal laser scanning microscopy (CLSM). The protoplast shape and structural changes of the ER were further examined after depolymerisation of actin microfilaments with latrunculin B (1 µM). In its natural state, the cortical ER is a dynamic network of fine tubes and cisternae underneath the plasma membrane. Under acute and long-term plasmolysis (up to 45 min), changes in the protoplast form and the cortical ER, as well as the formation of Hechtian strands and Hechtian reticula, were observed. The processing of the high-resolution z-scans allowed the creation of 3D models and gave detailed insight into the ER of living protonema cells before, during and after plasmolysis.

Fibronectin expression and organization in mesothelial and mesothelioma cells

1996 ◽  
Vol 271 (5) ◽  
pp. L804-L812 ◽  
Author(s):  
P. C. Ferriola ◽  
W. Stewart
Keyword(s):  
Laser Scanning ◽  
Neoplastic Cell ◽  
Northern Blotting ◽  
Mesothelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Neoplastic Cells ◽  
Protein Levels ◽  
Passage Number ◽  
Ribonuclease Protection

Mesothelial cells are believed to be the progenitor cells for malignant mesothelioma, a tumor associated with exposure to asbestos and other mineral fibers. Little is known regarding fibronectin (Fn) function in mesothelial and mesothelioma cells. Fn RNA, protein levels, and localization were assessed in secondary cultures and later passages of spontaneously immortalized rat pleural mesothelial (NRM) cells and in neoplastic cell lines derived from asbestos-induced mesotheliomas. NRM cells expressed similar levels of Fn RNA regardless of passage number or cell density, as determined by Northern blotting and ribonuclease protection assays. Western blotting showed that Fn protein was both secreted by NRM cells and associated with cell lysates. Immunofluorescent confocal laser scanning microscopy demonstrated that secondary cultures of NRM cells assembled Fn into abundant homogeneous fibrillar arrays organized primarily between cells, whereas later passages of NRM cells displayed abundant but less homogeneous Fn organization. Fn RNA and protein levels in neoplastic mesothelial cells were slightly less or similar to levels in NRM cells. Organization of Fn in neoplastic cells was heterogeneous compared with secondary cultures of NRM cells, but Fn fibril formation was still apparent. F-actin microfilaments were organized in both NRM and neoplastic cells; however, actin stress fibers were maintained in neoplastic cells, whereas NRM cells displayed dense actin peripheral bands at high density. The maintenance of organized Fn and actin in mesothelioma cells is surprising and may contribute to the localized growth and invasive properties of these tumors.

Immunocytochemical and immunoelectron microscopic localization of α-, β-, and γ-ENaC in rat kidney

2001 ◽  
Vol 280 (6) ◽  
pp. F1093-F1106 ◽  
Author(s):  
Henrik Hager ◽  
Tae-Hwan Kwon ◽  
Anna K. Vinnikova ◽  
Shyama Masilamani ◽  
Heddwen L. Brooks ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Subcellular Localization ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Medullary Collecting Duct ◽  
Epithelial Sodium Channel Enac

Epithelial sodium channel (ENaC) subunit (α, β, and γ) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. α-ENaC was localized mainly in a zone in the apical domains, whereas β- and γ-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, α-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, α-ENaC was present in a narrow zone near the apical plasma membrane, whereas β- and γ-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na+ reabsorption.

Human cytomegalovirus UL37 immediate-early regulatory proteins traffic through the secretory apparatus and to mitochondria

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1779-1789 ◽  
Author(s):  
Anamaris M. Colberg-Poley ◽  
Mital B. Patel ◽  
Darwin P. P. Erezo ◽  
Jay E. Slater
Keyword(s):  
Plasma Membrane ◽  
Human Cytomegalovirus ◽  
Laser Scanning ◽  
Human Cells ◽  
Laser Scanning Microscopy ◽  
Regulatory Proteins ◽  
Immediate Early ◽  
Confocal Laser ◽  
Amino Terminal ◽  
Secretory Apparatus

The human cytomegalovirus (HCMV) UL36–38 immediate-early (IE) locus encodes the UL37 exon 1 (pUL37x1) and UL37 (gpUL37) regulatory proteins, which have anti-apoptotic activities. pUL37x1 shares its entire sequence, including a hydrophobic leader and an acidic domain, with the exception of one residue, with the amino terminus of gpUL37. gpUL37 has, in addition, unique N-linked glycosylation, transmembrane and cytosolic domains. A rabbit polyvalent antiserum was generated against residues 27–40 in the shared amino-terminal domain and a mouse polyvalent antiserum was generated against the full-length protein to study trafficking of individual UL37 proteins in human cells that transiently expressed gpUL37 or pUL37x1. Co-localization studies by confocal laser scanning microscopy detected trafficking of gpUL37 and pUL37x1 from the endoplasmic reticulum to the Golgi apparatus in permissive U373 cells and in human diploid fibroblasts (HFF). Trafficking of gpUL37 to the cellular plasma membrane was detected in unfixed HFF cells. FLAG-tagged gpUL37 trafficked similarly through the secretory apparatus to the plasma membrane. By using confocal microscopy and immunoblotting of fractionated cells, gpUL37 and pUL37x1 were found to co-localize with mitochondria in human cells. This unconventional dual trafficking pattern through the secretory apparatus and to mitochondria is novel for herpesvirus IE regulatory proteins.

Low aquaporin-2 levels in polyuric DI +/+ severe mice with constitutively high cAMP-phosphodiesterase activity

1999 ◽  
Vol 276 (2) ◽  
pp. F179-F190 ◽  
Author(s):  
Jørgen Frøkiaer ◽  
David Marples ◽  
Heinz Valtin ◽  
John F. Morris ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Northern Blotting ◽  
Apical Plasma Membrane ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
Aquaporin 2 ◽  
Acute Response ◽  
Camp Levels

In the renal collecting duct, vasopressin acutely activates cAMP production, resulting in trafficking of aquaporin-2 water channels (AQP2) to the apical plasma membrane, thereby increasing water permeability. This acute response is modulated by long-term changes in AQP2 expression. Recently, a cAMP-responsive element has been identified in the AQP2 gene, raising the possibility that changes in cAMP levels may control AQP2 expression. To investigate this possibility, we determined AQP2 protein levels in a strain of mice, DI +/+ severe (DI), which have genetically high levels of cAMP-phosphodiesterase activity, and hence low cellular cAMP levels, and severe polyuria. Semiquantitative immunoblotting of membrane fractions prepared from whole kidneys revealed that AQP2 levels in DI mice were only 26 ± 7% (±SE) of those in control mice ( n = 10, P < 0.01). In addition, semiquantitative Northern blotting revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice compared with control mice (43 ± 6% vs. 100 ± 10%; n = 6 in each group, P < 0.05). AQP3 levels were also reduced. The mice were polyuric and urine osmolalities were accordingly substantially lower in the DI mice than in controls (496 ± 53 vs. 1,696 ± 105 mosmol/kgH2O, respectively). Moreover, there was a linear correlation between urine osmolalities and AQP2 levels ( P < 0.05). Immunoelectron microscopy confirmed the markedly lower expression of AQP2 in collecting duct principal cells in kidneys of DI mice and, furthermore, demonstrated that AQP2 was almost completely absent from the apical plasma membrane. Thus expression of AQP2 and AQP2 trafficking were severely impaired in DI mice. These results are consistent with the view that in vivo regulation of AQP2 expression by vasopressin is mediated by cAMP.

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