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Ultrastructure of the choroid plexus. In choroid plexus, the epithelial cell layer separates the brain ventricle (V) from the blood supply. The blood supply is found within the stroma (or the choroid) which contains blood vessels (BIQ and extracellular matrix. (A) The chick choroid plexus epithelium has a columnar morphology and numerous cilia on the apical surface. (B) Higher magnification of the chick choroid plexus epithelial cell apical-lateral junctional domain shows the tight and adherens junctions. (C) The rat choroid plexus epithelium has a more cuboidal morphology and a more extensive, dome-shaped apical domain, in contrast to that of the chick (A). (D) Higher magnification view of the rat choroid plexus epithelial cell junctional complex. Arrowheads indicate the tight junction, and arrows indicate the adherens junction. Bars: (A and C) 5/~m; (B and D) 1 /zm.
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In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion a...
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... the choroid plexus, a monolayer of epithelial cells separates the choroid blood supply from the brain ventricles (Fig. 2). In general, for both chick ( Fig. 2 A) and rat choroid plexus ( Fig. 2 C), the basal surface of the cells contacts a basement membrane that is adjacent to blood vessels and the stroma (extracellular matrix and fibroblasts). The apical membrane is directly exposed to the cerebrospinal fluid within the brain ventricles. ...
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... the choroid plexus, a monolayer of epithelial cells separates the choroid blood supply from the brain ventricles (Fig. 2). In general, for both chick ( Fig. 2 A) and rat choroid plexus ( Fig. 2 C), the basal surface of the cells contacts a basement membrane that is adjacent to blood vessels and the stroma (extracellular matrix and fibroblasts). The apical membrane is directly exposed to the cerebrospinal fluid within the brain ventricles. Characteristically, the apical sur- face has numerous, ...
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... the choroid plexus, a monolayer of epithelial cells separates the choroid blood supply from the brain ventricles (Fig. 2). In general, for both chick ( Fig. 2 A) and rat choroid plexus ( Fig. 2 C), the basal surface of the cells contacts a basement membrane that is adjacent to blood vessels and the stroma (extracellular matrix and fibroblasts). The apical membrane is directly exposed to the cerebrospinal fluid within the brain ventricles. Characteristically, the apical sur- face has numerous, swollen microvilli. A brush border ...
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... differences were observed between the rat and chick choroid plexus (Fig. 2, A and C). The rat choroid plexus is comprised of cuboidal cells, whereas the cells are columnar in the chick. Consequently, the lateral domain of the rat choroid plexus cells is short relative to that of the chick cells. Also, the rat choroid plexus epithelial cells have a more elaborate, dome-shaped apical membrane domain than that ...
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... immunoblots were probed with antisera specific for either the ix-or ~-subunit of Na+,K+-ATPase. Results showed that the slower migrating of the two protein bands reacted with the antibodies (Fig. 9, lanes I and 2). Analysis of immunoblots probed with antisera specific for either fo- drin or ankyrin revealed that both protein bands reacted (Fig. 9, lanes 3 and 5). In some experiments, we detected fodrin immunoreactivity in only the faster migrating of the two pro- tein bands (Fig. 9, lane 4); the reason for this difference is not known, but may ...
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... plasma membranes (Fig. 10), and that it colocalized with the membrane-cytoskeleton (Figs. 3, c and d and 4, c and d). Al- though cadherin staining was detected along the length of the lateral membrane, a greater intensity of cadherin staining was detected at the apex of the lateral membrane in the re- gion of the zonula adherens (compare with Fig. 2), similar to the distribution of cadherins in other epithelial cells (Boiler et al., 1985). Seventy six percent of cadherins were resistant to extraction from chick choroid plexus in buffers containing Triton X-100 (Fig. 5). This result is consistent with the possibility that cadherins expressed in the choroid plexus epithelium, like ...
Citations
... Thus, the transport of NH 4 + from the extrapallial fluid into the epithelial cells of the inner mantle may involve NKA, if it is located at the apical membrane of the shell-facing epithelium. However, NKA has a basolateral localization in nearly all types of epithelial cell [11]; the only exceptions are the choroid plexus [12], the retinal pigment epithelium [13] and the oral epithelium of the coral Acropora yongei [14] which express apical NKA. NKA has three types of subunits (α, β, and γ), and each type of subunit has its isoforms. ...
Na⁺/K⁺-ATPase (NKA) is essential for maintaining the Na⁺ and K⁺ gradients, and supporting the secondary active transport of certain ions/molecules, across the plasma membrane of animal cells. This study aimed to clone the NKA α-subunit (NKAα) from the inner mantle adjacent to the extrapallial fluid of Tridacna squamosa, to determine its subcellular localization, and to examine the effects of light exposure on its transcript level and protein abundance. The cDNA coding sequence of NKAα from T. squamosa comprised 3105 bp, encoding 1034 amino acids with an estimated molecular mass of 114 kDa. NKAα had a basolateral localization along the shell-facing epithelium of the inner mantle. Exposure to 12 h of light led to a significantly stronger basolateral NKAα-immunofluorescence at the shell-facing epithelium, indicating that NKA might play a role in light-enhanced calcification in T. squamosa. After 3 h of light exposure, the transcript level of NKAα decreased transiently in the inner mantle, but returned to the control level thereafter. In comparison, the protein abundance of NKAα remained unchanged at hour 3, but became significantly higher than the control after 12 h of light exposure. Hence, the expression of NKAα in the inner mantle of T. squamosa was light-dependent. It is probable that a higher expression level of NKA was needed in the shell-facing epithelial cells of the inner mantle to cope with a rise in Na⁺ influx, possibly caused by increases in activities of some Na⁺-dependent ion transporters/channels involved in light-enhanced calcification.
... Such mechanism was initially proposed for Na þ , K þ -ATPase in MDCK cells, in which a direct interaction of the pump's a subunit with ankyrin promotes its association with the spectrin cytoskeleton, which is concentrated laterally in MDCK cells (Morrow et al. 1989;Nelson and Hammerton 1989;Nelson et al. 1990). In epithelia where the Na þ , K þ -ATPase is apical, like the RPE and CP, this association with spectrin and ankyrin may contribute to the inverted polarized distribution of the pump (Gundersen et al. 1991;Marrs et al. 1993;Alper et al. 1994). However, whether these proteins form a complex has not been determined directly and the role played in determining polarized distribution is not clear because spectrin and Ankyrin may be also present at the basolateral membrane in CP (Marrs et al. 1993;Alper et al. 1994). ...
... In epithelia where the Na þ , K þ -ATPase is apical, like the RPE and CP, this association with spectrin and ankyrin may contribute to the inverted polarized distribution of the pump (Gundersen et al. 1991;Marrs et al. 1993;Alper et al. 1994). However, whether these proteins form a complex has not been determined directly and the role played in determining polarized distribution is not clear because spectrin and Ankyrin may be also present at the basolateral membrane in CP (Marrs et al. 1993;Alper et al. 1994). ...
Directional fluid flow is an essential process for embryo development as well as for organ and organism homeostasis. Here, we review the diverse structure of various organ-blood barriers, the driving forces, transporters, and polarity mechanisms that regulate fluid transport across them, focusing on kidney-, eye-, and brain-blood barriers. We end by discussing how cross talk between barrier epithelial and endothelial cells, perivascular cells, and basement membrane signaling contribute to generate and maintain organ-blood barriers.
... The following commercially available antibodies and dyes were used: β-catenin (mouse, 610154; BD); HA (mouse, HA.11, MMS-101P; Covance; rabbit, H6908; Sigma-Aldrich); Myc (mouse, 9E10, MMS- 150P; Covance); GAP DH (mouse, AB8245; Abcam); DEC MA (rat, U3254; Sigma-Aldrich); anti–human IgG Fc specific (goat, I2136; Sigma-Aldrich); RR1, mouse, which was generated in-house and recognizes the canine E-cadherin extracellular domain as has been described previously (Marrs et al., 1993); DαM568 (A10037, Thermo Fisher Scientific); Hoechst 33342 (H-3570; Molecular Probes); and Coomassie Brilliant Blue G-250 (20279; Thermo Fisher Scientific ). The rabbit αE-catenin antibody which recognizes full-length αE-catenin has been described previously (Näthke et al., 1994), and Alexa Fluor–conjugated secondary antibodies were used for immunofluorescence staining and immunoblotting. ...
As part of the E-cadherin-β-catenin-αE-catenin complex (CCC), mammalian αE-catenin binds F-actin weakly in the absence of force, whereas cytosolic αE-catenin forms a homodimer that interacts more strongly with F-actin. It has been concluded that cytosolic αE-catenin homodimer is not important for intercellular adhesion because E-cadherin/αE-catenin chimeras thought to mimic the CCC are sufficient to induce cell-cell adhesion. We show that, unlike αE-catenin in the CCC, these chimeras homodimerize, bind F-actin strongly, and inhibit the Arp2/3 complex, all of which are properties of the αE-catenin homodimer. To more accurately mimic the junctional CCC, we designed a constitutively monomeric chimera, and show that E-cadherin-dependent cell adhesion is weaker in cells expressing this chimera compared with cells in which αE-catenin homodimers are present. Our results demonstrate that E-cadherin/αE-catenin chimeras used previously do not mimic αE-catenin in the native CCC, and imply that both CCC-bound monomer and cytosolic homodimer αE-catenin are required for strong cell-cell adhesion.
... Main integral membrane protein of epithelial AJs; member of classical cadherin family (Type I) mediating homophilic adhesions in cis and trans; links to actin cytoskeleton via catenin complex (Vestweber, 2015) Basolateral localization in rat and chicken choroid plexus epithelium (Marrs et al., 1993) and epithelial cells of benign human choroid plexus tumors (Figarella-Branger et al., 1995) Core homophilic cell adhesion molecule of epithelial AJs, controls epithelial cell contact formation, barrier integrity, junctional plasticity, and cytoskeletal tension VE-cadherin (cadherin-5) ...
Unique intercellular junctional complexes between the central nervous system (CNS) microvascular endothelial cells and the choroid plexus epithelial cells form the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB), respectively. These barriers inhibit paracellular diffusion, thereby protecting the CNS from fluctuations in the blood. Studies of brain barrier integrity during development, normal physiology, and disease have focused on BBB and BCSFB tight junctions but not the corresponding endothelial and epithelial adherens junctions. The crosstalk between adherens junctions and tight junctions in maintaining barrier integrity is an understudied area that may represent a promising target for influencing brain barrier function.
© 2015 Tietz and Engelhardt.
... The majority of CSF is secreted by the choroid plexus (Nilsson et al. 1992). Water channel aquaporin-1 (AQP1; Bondy et al. 1993;Hasegawa et al. 1994;Nielsen et al. 1993) and Na + ,K + -ATPase (Marrs et al. 1993) occur together at the apical membrane of the choroidal epithelial cells and seem to be involved in CSF secretion. Several studies have indicated that ependymal cells, which overlie the entire ventricular system, also participate in the maintenance of these fluids. ...
Brain ependymal cells, which form an epithelial layer covering the cerebral ventricles, have been shown to play a role in the regulation of cerebrospinal and interstitial fluids. The machinery underlying this, however, remains largely unknown. Here, we report the specific localization of an inwardly rectifying K(+) channel, Kir4.1, on the ependymal cell membrane suggesting involvement of the channel in this function. Immunohistochemical study with confocal microscopy identified Kir4.1 labeling on the lateral but not apical membrane of ependymal cells. Ultrastructural analysis revealed that Kir4.1-immunogold particles were specifically localized and clustered on adjacent membranes at puncta adherens type junctions, whereas an aquaporin water channel, AQP4, that was also detected on the lateral membrane only occurred at components other than adherens junctions. Therefore, in ependymal cells, Kir4.1 and AQP4 are partitioned into distinct membrane compartments that might respectively transport either K(+) or water. Kir4.1 was also expressed in a specialized form of ependymal cell, namely the tanycyte, being abundant in tanycyte processes wrapping neuropils and blood vessels. These specific localizations suggest that Kir4.1 mediates intercellular K(+) exchange between ependymal cells and also K(+)-buffering transport via tanycytes that can interconnect neurons and vessels/ventricles. We propose that ependymal cells and tanycytes differentially operate Kir4.1 and AQP4 actively to control the property of fluids at local areas in the brain.
... 123,124 Conversely, ankyrin and fodrin were both found at the apical surface of choroid plexus and RPE cells, where ankyrin directly interacts with Na, K-ATPase. 125,126 Fodrin and ankyrin also localize to the lateral membrane of choroid plexus cells, however, 125 suggesting that the presence of these cytoskeletal linkers is not sufficient to specify Na,K-ATPase targeting. It is more likely that this cytoskeletal attachment plays a role in retention at the membrane, rather than directly determining the pump's initial trafficking or ultimate distribution. ...
... 123,124 Conversely, ankyrin and fodrin were both found at the apical surface of choroid plexus and RPE cells, where ankyrin directly interacts with Na, K-ATPase. 125,126 Fodrin and ankyrin also localize to the lateral membrane of choroid plexus cells, however, 125 suggesting that the presence of these cytoskeletal linkers is not sufficient to specify Na,K-ATPase targeting. It is more likely that this cytoskeletal attachment plays a role in retention at the membrane, rather than directly determining the pump's initial trafficking or ultimate distribution. ...
Renal epithelial cells must maintain distinct protein compositions in their apical and basolateral membranes in order to perform their transport functions. The creation of these polarized protein distributions depends on sorting signals that designate the trafficking route and site of ultimate functional residence for each protein. Segregation of newly synthesized apical and basolateral proteins into distinct carrier vesicles can occur at the trans-Golgi network, recycling endosomes, or a growing assortment of stations along the cellular trafficking pathway. The nature of the specific sorting signal and the mechanism through which it is interpreted can influence the route a protein takes through the cell. Cell type-specific variations in the targeting motifs of a protein, as are evident for Na,K-ATPase, demonstrate a remarkable capacity to adapt sorting pathways to different developmental states or physiologic requirements. This review summarizes our current understanding of apical and basolateral trafficking routes in polarized epithelial cells.
... In most epithelia, the Na + ,K + -ATPase is linked to the spectrin cytoskeleton through ankyrins and thus, all these proteins accumulate in the basolateral cell domain (Morrow et al., 1989; Nelson and Hammerton, 1989 ). In the CPE, the general spectrin cytoskeleton and undefined ankyrins are found primarily near the luminal membrane as opposed to most other polarized epithelia (Marrs et al., 1993; Alper et al., 1994 ). It is unknown whether the atypical distribution of the membrane proteins in the CPE is caused by cell type specific distribution of cytoskeletal proteins, anchoring proteins, membrane proteins, or other factors. ...
... Adducins are alternatives to ankyrins for linking AE2 to the cytoskeleton at the basolateral membrane of the choroid plexus, as they co-sediment separately from ankyrin and the Na + ,K + - ATPase in sucrose gradients (Marrs et al., 1993). Adducins bind both the spectrin and actin cytoskeleton and adducin immunoreactivity was previously shown in proximity to AE2 in the CPE (Alper et al., 1994 ). ...
... The Na + ,K + -ATPase is linked to the spectrin cytoskeleton through ankyrin-3 in the choroid plexus (Marrs et al., 1993). The expression of α1-Na + ,K + -ATPase was greatly decreased in slc4a10 ko mice (Damkier and Praetorius, 2012). ...
The choroid plexus epithelium (CPE) has served as a model-epithelium for cell polarization and transport studies and plays a crucial role for cerebrospinal fluid (CSF) production. The normal luminal membrane expression of Na⁺,K⁺-ATPase, aquaporin-1 and Na⁺/H⁺ exchanger 1 in the choroid plexus is severely affected by deletion of the slc4a10 gene that encodes the bicarbonate transporting protein Ncbe/NBCn2. The causes for these deviations from normal epithelial polarization and redistribution following specific gene knockout are unknown, but may be significant for basic epithelial cell biology. Therefore, a more comprehensive analysis of cell polarization in the choroid plexus is warranted. We find that the cytoskeleton in the choroid plexus contains αI-, αII-, βI-, and βII-spectrin isoforms along with the anchoring protein ankyrin-3, most of which are mainly localized in the luminal membrane domain. Furthermore, we find α-adducin localized near the plasma membranes globally, but with only faint expression in the luminal membrane domain. In slc4a10 knockout mice, the abundance of β1 Na⁺,K⁺-ATPase subunits in the luminal membrane is markedly reduced. Anion exchanger 2 abundance is increased in slc4a10 knockout and its anchor protein, α-adducin is almost exclusively found near the basolateral domain. The αI- and βI-spectrin abundances are also decreased in the slc4a10 knockout, where the basolateral domain expression of αI-spectrin is exchanged for a strictly luminal domain localization. E-cadherin expression is unchanged in the slc4a10 knockout, while small decreases in abundance are observed for its probable adaptor proteins, the catenins. Interestingly, the abundance of the tight junction protein claudin-2 is significantly reduced in the slc4a10 knockouts, which may critically affect paracellular transport in this epithelium. The observations allow the generation of new hypotheses on basic cell biological paradigms that can be tested experimentally in future studies.
... The earliest mechanistic insight into the atypical distribution of membrane proteins in the choroid plexus came from two studies published in the early 1990s. In the first, Marrs et al. (200) found that the Na ϩ -K ϩ -ATPase was complexed by erythoroid ankyrin and fodrin to the luminal surface in a triton-resistant membrane protein fraction. The now accepted nomenclature defines the erythroid ankyrin A as ankyrin 1, whereas the former brain (and cardiac) ankyrin B is ankyrin 2 and cardiac/neuronal ankyrin G is now ankyrin 3. Fodrin is a nonerythroid spectrin heterodimer composed of ␣II and II subunits (erythroid type is ␣I and I). ...
... In agreement with the study by Marrs et al. (200), spectrin was also found at the basolateral domain but in a much lower abundance. However, the immunohistochemical localization of ankyrin with the luminal membrane in the Alper study (5) was in stark contrast to the biochemical detection of ankyrin in both domains of the cell by Marrs et al. (200). Apart from the methodological difference, this discrepancy may have been caused by differences in the ankyrin subtype or antibody performance between the two studies. ...
... Apart from the methodological difference, this discrepancy may have been caused by differences in the ankyrin subtype or antibody performance between the two studies. In Alper's study (5), the antibodies were directed against human erythroid ankyrin (ANK1), while they were anti-chicken ANK1 in the Marrs study (200). It was recently shown that the subluminal ezrin distribution in the CPE was completely disrupted in mice with genetic ablation of the NaHCO 3 transporter Ncbe (59). ...
The choroid plexus epithelium is a cuboidal cell monolayer, which produces the majority of the cerebrospinal fluid. The concerted action of a variety of integral membrane proteins mediates the transepithelial movement of solutes and water across the epithelium. Secretion by the choroid plexus is characterized by an extremely high rate and by the unusual cellular polarization of well-known epithelial transport proteins. This review focuses on the specific ion and water transport by the choroid plexus cells, and then attempts to integrate the action of specific transport proteins to formulate a model of cerebrospinal fluid secretion. Significant emphasis is placed on the concept of isotonic fluid transport across epithelia, as there is still surprisingly little consensus on the basic biophysics of this phenomenon. The role of the choroid plexus in the regulation of fluid and electrolyte balance in the central nervous system is discussed, and choroid plexus dysfunctions are described in a very diverse set of clinical conditions such as aging, Alzheimer's disease, brain edema, neoplasms, and hydrocephalus. Although the choroid plexus may only have an indirect influence on the pathogenesis of these conditions, the ability to modify epithelial function may be an important component of future therapies.
... RFP containing protein chimeras were immunoprecipitated using a rabbit polyclonal RFP antibody at 1:100 dilution (Clonetech; catalog # 632397). Ecadherin immunoprecipitations used E2 antibody raised against the cytoplasmic domain of E-cadherin [26]. Cells were incubated with 10 mM MG-132 for 4 hours prior to extraction. 100 mM cycloheximide (CHX) was added to cell culture media for up to 6 hours prior to extraction. ...
p120-Catenin binding to, and Hakai-mediated ubiquitination of the E-cadherin juxtamembrane domain (JMD) are thought to be involved in regulating E-cadherin internalization and degradation. However, the relationship between these two pathways is not understood. We targeted the E-cadherin JMD to mitochondria (WT-JMD) to isolate this domain from the plasma membrane and internalization, and to examine protein modifications and degradation. WT-JMD localized to mitochondria, but did not accumulate there except when proteasome activity was inhibited. We found WT-JMD was ubiquitinated, and arginine substitution of lysines at position 5 (K5R) and 83 (K83R) resulted in the stable accumulation of mutant JMD at mitochondria. p120-Catenin did not localize, or bind to WT-JMD even upon proteasome inhibition, whereas the K5,83R-JMD mutant bound and localized p120-catenin to mitochondria. Mutation of the p120-catenin binding site in combination with these lysine mutations inhibited p120-catenin binding, but did not decrease JMD stability or its accumulation at mitochondria. Thus, increased stability of JMD lysine mutants was due to inhibition of ubiquitination and not to p120-catenin binding. Finally, mutation of these critical lysines in full length E-cadherin had similar effects on protein stability as WT-JMD. Our results indicate that ubiquitination of the JMD inhibits p120-catenin binding, and targets E-cadherin for degradation.
... A RalB mAb was a generous gift from Michael White (University of Texas Southwestern Medical Center, Dallas, TX). A rabbit polyclonal antibody against Ecadherin was a generous gift from W. James Nelson (Stanford University, Stanford, CA; Marrs et al ., 1993 ). Fluorescein isothiocyanate (FITC)-goat anti-mouse and Texas red-donkey anti-rabbit immunoglobulin G (IgG) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). ...
Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference-mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical-basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.
