scholarly journals Normal Establishment of Epithelial Tight Junctions in Mice and Cultured Cells Lacking Expression of ZO-3, a Tight-Junction MAGUK Protein

2006 ◽  
Vol 26 (23) ◽  
pp. 9003-9015 ◽  
Author(s):  
Makoto Adachi ◽  
Akihito Inoko ◽  
Masaki Hata ◽  
Kyoko Furuse ◽  
Kazuaki Umeda ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Cultured Cells ◽  
Molecular Architecture ◽  
Wild Type ◽  
Teratocarcinoma Cell ◽  
Cytoplasmic Surface ◽  
Teratocarcinoma Cells ◽  
F9 Teratocarcinoma ◽  
Maguk Protein

ABSTRACT ZO-1, ZO-2, and ZO-3 are closely related MAGUK family proteins that localize at the cytoplasmic surface of tight junctions (TJs). ZO-1 and ZO-2 are expressed in both epithelia and endothelia, whereas ZO-3 is exclusively expressed in epithelia. In spite of intensive studies of these TJ MAGUKs, our knowledge of their functions in vivo, especially those of ZO-3, is still fragmentary. Here, we have generated mice, as well as F9 teratocarcinoma cell lines, that do not express ZO-3 by homologous recombination. Unexpectedly, ZO-3 −/− mice were viable and fertile, and rigorous phenotypic analyses identified no significant abnormalities. Moreover, ZO-3-deficient F9 teratocarcinoma cells differentiated normally into visceral endoderm epithelium-like cells in the presence of retinoic acid. These cells had a normal epithelial appearance, and the molecular architecture of their TJs did not appear to be affected, except that TJ localization of ZO-2 was upregulated. Suppression of ZO-2 expression by RNA interference in ZO-3 −/− cells, however, did not affect the architecture of TJs. Furthermore, the speed with which TJs formed after a Ca2+ switch was indistinguishable between wild-type and ZO-3 −/− cells. These findings indicate that ZO-3 is dispensable in vivo in terms of individual viability, epithelial differentiation, and the establishment of TJs, at least in the laboratory environment.

scholarly journals Nano-scale architecture of blood-brain barrier tight-junctions

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Esther Sasson ◽  
Shira Anzi ◽  
Batia Bell ◽  
Oren Yakovian ◽  
Meshi Zorsky ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Blood Brain Barrier ◽  
Super Resolution ◽  
Brain Barrier ◽  
Molecular Architecture ◽  
Mouse Development ◽  
Nano Scale ◽  
Blood Brain ◽  
Structural And Functional Properties

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in-vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery.

scholarly journals Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

2001 ◽  
Vol 12 (9) ◽  
pp. 2894-2905 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Mark A. McNiven
Keyword(s):  
Cultured Cells ◽  
Immunogold Labeling ◽  
Wild Type ◽  
Tubule Formation ◽  
Defective Mutant ◽  
Ring Structures ◽  
Membrane Tubules ◽  
Large Gtpases

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5′-O-(3-thio)triphosphate (GTPγS) but not GTP. EM of the GTPγS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPγS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

scholarly journals Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways

2018 ◽  
Vol 29 (6) ◽  
pp. 1720-1730 ◽  
Author(s):  
Miriam Zacchia ◽  
Xuefei Tian ◽  
Enrica Zona ◽  
Robert J. Alpern ◽  
Patricia A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Signaling Pathways ◽  
Cultured Cells ◽  
Wild Type ◽  
Experimental Conditions ◽  
Opossum Kidney ◽  
Citrate Transporter ◽  
Stimulation Of

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2−/−) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro. Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2−/− mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro. Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

scholarly journals Groucho Oligomerization Is Required for Repression In Vivo

2004 ◽  
Vol 24 (10) ◽  
pp. 4341-4350 ◽  
Author(s):  
Haiyun Song ◽  
Peleg Hasson ◽  
Ze’ev Paroush ◽  
Albert J. Courey
Keyword(s):  
Target Genes ◽  
Cultured Cells ◽  
Coiled Coil ◽  
S2 Cells ◽  
Wild Type ◽  
Developmental Defects ◽  
Conserved Domain ◽  
Wing Disk ◽  
Single Motif

ABSTRACT Drosophila Groucho (Gro) is a member of a family of metazoan corepressors with widespread roles in development. Previous studies indicated that a conserved domain in Gro, termed the Q domain, was required for repression in cultured cells and mediated homotetramerization. Evidence presented here suggests that the Q domain contains two coiled-coil motifs required for oligomerization and repression in vivo. Mutagenesis of the putative hydrophobic faces of these motifs, but not of the hydrophilic faces, prevents the formation of both tetramers and higher order oligomers. Mutagenesis of the hydrophobic faces of both coiled-coil motifs in the context of a Gal4-Gro fusion protein prevents repression of a Gal4-responsive reporter in S2 cells, while mutagenesis of a single motif weakens repression. The finding that the repression directed by the single mutants depends on endogenous wild-type Gro further supports the idea that oligomerization plays a role in repression. Overexpression in the fly of forms of Gro able to oligomerize, but not of a form of Gro unable to oligomerize, results in developmental defects and ectopic repression of Gro target genes in the wing disk. Although the function of several corepressors is suspected to involve oligomerization, these studies represent one of the first direct links between corepressor oligomerization and repression in vivo.

scholarly journals Herpes Simplex Virus with Highly Reduced gD Levels Can Efficiently Enter and Spread between Human Keratinocytes

2001 ◽  
Vol 75 (21) ◽  
pp. 10309-10318 ◽  
Author(s):  
Mary T. Huber ◽  
Todd W. Wisner ◽  
Nagendra R. Hegde ◽  
Kimberley A. Goldsmith ◽  
Daniel A. Rauch ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cultured Cells ◽  
Human Keratinocytes ◽  
Hacat Cells ◽  
Wild Type ◽  
Simplex Virus ◽  
Cell Spread ◽  
Hsv 1

ABSTRACT The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.

scholarly journals Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

1997 ◽  
Vol 17 (10) ◽  
pp. 5679-5687 ◽  
Author(s):  
C P Chang ◽  
Y Jacobs ◽  
T Nakamura ◽  
N A Jenkins ◽  
N G Copeland ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Hox Proteins ◽  
Amino Terminal ◽  
Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

scholarly journals Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells

2010 ◽  
Vol 298 (4) ◽  
pp. F935-F940 ◽  
Author(s):  
Janet D. Klein ◽  
Mitsi A. Blount ◽  
Otto Fröhlich ◽  
Chad E. Denson ◽  
Xiaoxiao Tan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Cell Lines ◽  
Specific Antibody ◽  
Cultured Cells ◽  
Apical Plasma Membrane ◽  
Wild Type ◽  
Amino Acid Peptide ◽  
In The Wild

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-A1 urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-A1 phosphorylation at S486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed S486 in roughly the center of the sequence. We also developed two stably transfected mIMCD3 cell lines: one expressing wild-type UT-A1 and one expressing a mutated form of UT-A1, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. The phospho-S486-UT-A1 antibody identified UT-A1 protein in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospho-S486-UT-A1 (measured using the phospho-S486 antibody) and of total UT-A1 phosphorylation (measured by 32P incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-A1 in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-A1 appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-A1 and an S486-UT-A1 phospho-specific antibody. We confirmed that vasopressin increases UT-A1 accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-A1 at S486 in rat IMCDs and that the S486-phospho-UT-A1 form is primarily detected in the apical plasma membrane.

scholarly journals Lysine methylation represses p53 activity in teratocarcinoma cancer cells

2016 ◽  
Vol 113 (35) ◽  
pp. 9822-9827 ◽  
Author(s):  
Jiajun Zhu ◽  
Zhixun Dou ◽  
Morgan A. Sammons ◽  
Arnold J. Levine ◽  
Shelley L. Berger
Keyword(s):  
Cancer Cells ◽  
Transcriptional Activity ◽  
P53 Protein ◽  
Lysine Methylation ◽  
Wild Type ◽  
Teratocarcinoma Cell ◽  
Teratocarcinoma Cells ◽  
Elevated Expression ◽  
Wild Type P53 ◽  
Tumor Suppressive Function

TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53’s transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma.

scholarly journals Androgen Receptor Acetylation Governs trans Activation and MEKK1-Induced Apoptosis without Affecting In Vitro Sumoylation and trans-Repression Function

2002 ◽  
Vol 22 (10) ◽  
pp. 3373-3388 ◽  
Author(s):  
Maofu Fu ◽  
Chenguang Wang ◽  
Jian Wang ◽  
Xueping Zhang ◽  
Toshiyuki Sakamaki ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Cultured Cells ◽  
Nuclear Hormone Receptor ◽  
Dependent Manner ◽  
Wild Type ◽  
Acetylation Site ◽  
Induced Apoptosis

ABSTRACT The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-κB, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.

scholarly journals Arenavirus Genome Rearrangement for the Development of Live Attenuated Vaccines

2015 ◽  
Vol 89 (14) ◽  
pp. 7373-7384 ◽  
Author(s):  
Benson Yee Hin Cheng ◽  
Emilio Ortiz-Riaño ◽  
Juan Carlos de la Torre ◽  
Luis Martínez-Sobrido
Keyword(s):  
Cultured Cells ◽  
Reporter Genes ◽  
Hemorrhagic Fever ◽  
Off Label Use ◽  
Wild Type ◽  
Lethal Challenge ◽  
Live Attenuated Vaccines ◽  
Expression Levels ◽  
Off Label

ABSTRACTSeveral members of theArenaviridaefamily cause hemorrhagic fever disease in humans and pose serious public health problems in their geographic regions of endemicity as well as a credible biodefense threat. To date, there have been no FDA-approved arenavirus vaccines, and current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Arenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome. Each genome segment uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientations, separated by a noncoding intergenic region. Here we have used minigenome-based approaches to evaluate expression levels of reporter genes from the nucleoprotein (NP) and glycoprotein precursor (GPC) loci within the S segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). We found that reporter genes are expressed to higher levels from the NP than from the GPC locus. Differences in reporter gene expression levels from the NP and GPC loci were confirmed with recombinant trisegmented LCM viruses. We then used reverse genetics to rescue a recombinant LCMV (rLCMV) containing a translocated viral S segment (rLCMV/TransS), where the viral NP and GPC open reading frames replaced one another. The rLCMV/TransS showed slower growth kinetics in cultured cells and was highly attenuatedin vivoin a mouse model of lethal LCMV infection, but immunization with rLCMV/TransS conferred complete protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV/TransS was associated with reduced NP expression levels. These results open a new avenue for the development of arenavirus live attenuated vaccines based on rearrangement of their viral genome.IMPORTANCESeveral arenaviruses cause severe hemorrhagic fever in humans and also pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections and antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and associated with side effects. Here we describe, for the first time, the generation of a recombinant LCMV where the viral protein products encoded by the S RNA segment (NP and GPC) were swapped to generate rLCMV/TransS. rLCMV/TransS exhibited reduced viral multiplication in cultured cells and was highly attenuatedin vivowhile conferring protection, upon a single immunization dose, against a lethal challenge with wild-type LCMV. Our studies provide a proof of concept for the rational development of safe and protective live attenuated vaccine candidates based on genome reorganization for the treatment of pathogenic arenavirus infections in humans.

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