scholarly journals Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells

2018 ◽  
Vol 315 (2) ◽  
pp. C258-C276 ◽  
Author(s):  
Rainelli Koumangoye ◽  
Salma Omer ◽  
Eric Delpire
Keyword(s):  
Epithelial Cells ◽  
De Novo ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
De Novo Mutation ◽  
Wild Type ◽  
Saliva Secretion

We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na+-K+-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.

scholarly journals Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

1996 ◽  
Vol 135 (1) ◽  
pp. 153-167 ◽  
Author(s):  
J Peränen ◽  
P Auvinen ◽  
H Virta ◽  
R Wepf ◽  
K Simons
Keyword(s):  
Epithelial Cells ◽  
Membrane Transport ◽  
Vesicular Stomatitis Virus ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Wild Type ◽  
Expression Systems ◽  
Vesicular Stomatitis

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

Identification of a novel signal in the cytoplasmic tail of the Na+:HCO3− cotransporter NBC1 that mediates basolateral targeting

2007 ◽  
Vol 292 (4) ◽  
pp. F1245-F1255 ◽  
Author(s):  
Hong C. Li ◽  
Emily Y. Li ◽  
Lisa Neumeier ◽  
Laura Conforti ◽  
Manoocher Soleimani
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Cytoplasmic Tail ◽  
Membrane Targeting ◽  
Wild Type ◽  
Functional Studies ◽  
Basolateral Targeting

The Na+:HCO3− cotransporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010–1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Furthermore, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins.

scholarly journals Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 3081-3093 ◽  
Author(s):  
Cunxi Li ◽  
Mingming Hao ◽  
Zheng Cao ◽  
Wei Ding ◽  
Ramona Graves-Deal ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Distinct Region ◽  
Lateral Membrane ◽  
Polarized Epithelial Cells

Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-α and that TGF-α is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of μ1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α–containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.

scholarly journals Human syntaxin 3 is localized apically in human intestinal cells

1997 ◽  
Vol 110 (18) ◽  
pp. 2207-2214 ◽  
Author(s):  
M.H. Delgrossi ◽  
L. Breuza ◽  
C. Mirre ◽  
P. Chavrier ◽  
A. Le Bivic
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Intestinal Epithelial ◽  
Human Intestinal Epithelial Cells ◽  
Membrane Components ◽  
Syntaxin 3 ◽  
Transgenic Drosophila

To understand the molecular mechanisms underlying sorting of apical and basolateral membrane components in human intestinal epithelial cells, we have cloned the human homolog of rat syntaxin 3 and looked for its subcellular localization. Endogenous human syntaxin 3 was found to be localized at the apical membrane of colon epithelial and Caco-2 cells. This apical localization was confirmed by confocal microscopy after transfection of the cDNA coding for either full length or N-terminally truncated human syntaxin 3 in Caco-2 cells. Furthermore the signal(s) and machinery targeting human syntaxin 3 to the apical membrane of epithelial cells are conserved between species since human syntaxin 3 was also localized at the apical membrane of canine MDCK cells and of epithelial cells in transgenic Drosophila melanogaster.

scholarly journals The NH2-terminus of Norepinephrine Transporter Contains a Basolateral Localization Signal for Epithelial Cells

2001 ◽  
Vol 12 (12) ◽  
pp. 3797-3807 ◽  
Author(s):  
Howard H. Gu ◽  
Xiaohong Wu ◽  
Bruno Giros ◽  
Marc G. Caron ◽  
Michael J. Caplan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Chimeric Protein ◽  
Norepinephrine Transporter ◽  
Apical Plasma Membrane ◽  
Wild Type ◽  
Localization Signal ◽  
Apical Localization

When expressed in epithelial cells, dopamine transporter (DAT) was detected predominantly in the apical plasma membrane, whereas norepinephrine transporter (NET) was found in the basolateral membrane, despite 67% overall amino acid sequence identity. To identify possible localization signals responsible for this difference, DAT–NET chimeras were expressed in MDCK cells and localized by immunocytochemistry and transport assays. The results suggested that localization of these transporters in MDCK cells depends on their highly divergent NH2-terminal regions. Deletion of the first 58 amino acids of DAT (preceding TM1) did not change its apical localization. However, the replacement of that region with corresponding sequence from NET resulted in localization of the chimeric protein to the basolateral membrane, suggesting that the NH2-terminus of NET, which contains two dileucine motifs, contains a basolateral localization signal. Mutation of these leucines to alanines in the context of a basolaterally localized NET/DAT chimera restored transporter localization to the apical membrane, indicating that the dileucine motifs are critical to the basolateral localization signal embodied within the NET NH2-terminal region. However, the same mutation in the context of wild-type NET did not disrupt basolateral localization, indicating the presence of additional signals in NET directing its basolateral localization within the plasma membrane.

Luminal flow rate regulates proximal tubule H-HCO3 transporters

1992 ◽  
Vol 262 (1) ◽  
pp. F47-F54 ◽  
Author(s):  
P. A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Flow Rate ◽  
Initial Rate ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Rate Dependence ◽  
Unstirred Layer ◽  
Disulfonic Acid ◽  
Luminal Flow

In vivo microperfusion was used to examine the mechanism of luminal flow rate dependence of proximal tubule acidification. Luminal flow rate was acutely changed between 5 and 40 nl/min, while luminal and peritubular capillary composition were held constant. With inhibition of basolateral membrane base transport by peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), cell pH (pHi) provides a sensitive index of apical membrane H secretory activity. At a luminal perfusate [HCO3] of 25 mM, progressive increases in luminal flow rate (5----15----25----40 nl/min) caused progressive increases in pHi. This effect was of a smaller magnitude with a luminal perfusate [HCO3] of 60 mM and was further decreased at a luminal perfusate [HCO3] of 100 mM. This pattern of diminished flow rate dependence at higher luminal [HCO3] is consistent with the presence of a luminal unstirred layer, whose composition can be modified by luminal flow rate. The activity of the apical membrane Na-H antiporter, assayed as the initial rate of pHi recovery from an acid load in the presence of peritubular DIDS, was faster at 40 compared with 5 nl/min. Basolateral membrane Na-3HCO3 symporter activity, assayed as the initial rate of pHi recovery from an alkali load in the absence of luminal and peritubular chloride, was faster at 40 compared with 5 nl/min. This effect was eliminated by luminal amiloride, suggesting an indirect effect of flow mediated by changes in pHi secondary to flow rate-dependent changes in apical membrane Na-H antiporter activity. In summary, increases in luminal flow rate directly increase apical membrane H secretion, possibly by modification of a luminal unstirred layer.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

2001 ◽  
Vol 152 (6) ◽  
pp. 1183-1196 ◽  
Author(s):  
Atsushi Suzuki ◽  
Tomoyuki Yamanaka ◽  
Tomonori Hirose ◽  
Naoyuki Manabe ◽  
Keiko Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Protein Complex ◽  
Mdck Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Surface Polarity ◽  
C Elegans ◽  
Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

scholarly journals Expression of lysophosphatidic acid receptor 5 is necessary for the regulation of intestinal Na+/H+ exchanger 3 by lysophosphatidic acid in vivo

2018 ◽  
Vol 315 (4) ◽  
pp. G433-G442 ◽  
Author(s):  
Kayte A. Jenkin ◽  
Peijian He ◽  
C. Chris Yun
Keyword(s):  
Epithelial Cells ◽  
Lysophosphatidic Acid ◽  
Direct Evidence ◽  
Intestinal Epithelial Cells ◽  
Regulatory Role ◽  
Intestinal Epithelial ◽  
Fluid Absorption ◽  
Wild Type

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA5 in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC. Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5.

scholarly journals Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells

1992 ◽  
Vol 103 (3) ◽  
pp. 677-687 ◽  
Author(s):  
G.K. Ojakian ◽  
R. Schwimmer
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Transport Pathways ◽  
Intracellular Targeting ◽  
Polarized Delivery ◽  
Membrane Recognition

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

scholarly journals Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament in vivo

2016 ◽  
Author(s):  
Maureen M. Mundia ◽  
Alissa C. Magwood ◽  
Mark D. Baker
Keyword(s):  
Homologous Recombination ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Atp Hydrolysis ◽  
Dominant Negative ◽  
Strand Exchange ◽  
Wild Type ◽  
Hybridoma Cell Lines ◽  
Insight Into

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

Sign in / Sign up

Export Citation Format

Share Document