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Polarization of membrane associated proteins in the choroid plexus epithelium from normal and slc4a10 knockout mice.

Christensen IB, Gyldenholm T, Damkier HH, Praetorius J - Front Physiol (2013)

Bottom Line: Anion exchanger 2 abundance is increased in slc4a10 knockout and its anchor protein, α-adducin is almost exclusively found near the basolateral domain.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, Faculty of Health, Aarhus University Aarhus, Denmark.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Ezrin and moesin expression in CPE from slc4a10 wt and ko mice. Mouse brain sections were immunofluorescence stained with ezrin and moesin antibodies (in green). (A) Immunohistochemical detection of ezrin in slc4a10 wt and ko, as indicated. Bar graph on the right show the semi-quantitation of the ezrin immunofluorescence in slc4a10 wt and ko mouse CPE (wt: n = 5, ko: n = 4). (B) Immunohistochemical detection of ezrin at a higher magnification in slc4a10 wt and ko, as indicated. (C) Immunostaining for moesin in slc4a10 wt and ko, respectively. Bar graph on the right show the semi-quantitation of the moesin immunofluorescence in slc4a10 wt and ko mouse CPE (wt: n = 5, ko: n = 4). Arrows indicate the luminal membrane, while arrowheads indicate the basolateral membrane. Cell nuclei were visualized by Topro nuclear staining (blue).
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Figure 4: Ezrin and moesin expression in CPE from slc4a10 wt and ko mice. Mouse brain sections were immunofluorescence stained with ezrin and moesin antibodies (in green). (A) Immunohistochemical detection of ezrin in slc4a10 wt and ko, as indicated. Bar graph on the right show the semi-quantitation of the ezrin immunofluorescence in slc4a10 wt and ko mouse CPE (wt: n = 5, ko: n = 4). (B) Immunohistochemical detection of ezrin at a higher magnification in slc4a10 wt and ko, as indicated. (C) Immunostaining for moesin in slc4a10 wt and ko, respectively. Bar graph on the right show the semi-quantitation of the moesin immunofluorescence in slc4a10 wt and ko mouse CPE (wt: n = 5, ko: n = 4). Arrows indicate the luminal membrane, while arrowheads indicate the basolateral membrane. Cell nuclei were visualized by Topro nuclear staining (blue).

Mentions: Previously, we have characterized the slc4a10 ko mouse (Jacobs et al., 2008) and showed that the cellular localization of NHE1 in these mice was found in the basolateral plasma membrane instead of in the luminal membrane as in the slc4a10 wt mouse (Damkier et al., 2009). Therefore, antibodies reacting specifically against the ezrin and moesin proteins known to interact with NHE1 directly or indirectly were used for immunohistochemical analysis of CPE. As previously observed, ezrin staining is found in the cytoplasm close to the luminal plasma membrane in the wt CPE (Figure 4A, micrographs). Ezrin staining was more variable among slc4a10 ko mice, but was also here found predominantly in the subluminal domain in the CPE as assessed by immunofluorescence (Figure 4B). Figure 5 shows that the ezrin distribution appears more cytosolic in slc4a10 ko mice compared to slc4a10 wt in peroxidase stained sections from the same mice. Some labeling is observed in the luminal membrane domain, but the labeling is also found in intracellular compartments. The bar graph in Figure 4A shows that the semi-quantified immunofluorescence signal for ezrin did not differ between slc4a10 wt and ko mice, (p = 0.556, n = 5 and 4 for wt and ko, respectively).


Polarization of membrane associated proteins in the choroid plexus epithelium from normal and slc4a10 knockout mice.

Christensen IB, Gyldenholm T, Damkier HH, Praetorius J - Front Physiol (2013)

Ezrin and moesin expression in CPE from slc4a10 wt and ko mice. Mouse brain sections were immunofluorescence stained with ezrin and moesin antibodies (in green). (A) Immunohistochemical detection of ezrin in slc4a10 wt and ko, as indicated. Bar graph on the right show the semi-quantitation of the ezrin immunofluorescence in slc4a10 wt and ko mouse CPE (wt: n = 5, ko: n = 4). (B) Immunohistochemical detection of ezrin at a higher magnification in slc4a10 wt and ko, as indicated. (C) Immunostaining for moesin in slc4a10 wt and ko, respectively. Bar graph on the right show the semi-quantitation of the moesin immunofluorescence in slc4a10 wt and ko mouse CPE (wt: n = 5, ko: n = 4). Arrows indicate the luminal membrane, while arrowheads indicate the basolateral membrane. Cell nuclei were visualized by Topro nuclear staining (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842056&req=5

Figure 4: Ezrin and moesin expression in CPE from slc4a10 wt and ko mice. Mouse brain sections were immunofluorescence stained with ezrin and moesin antibodies (in green). (A) Immunohistochemical detection of ezrin in slc4a10 wt and ko, as indicated. Bar graph on the right show the semi-quantitation of the ezrin immunofluorescence in slc4a10 wt and ko mouse CPE (wt: n = 5, ko: n = 4). (B) Immunohistochemical detection of ezrin at a higher magnification in slc4a10 wt and ko, as indicated. (C) Immunostaining for moesin in slc4a10 wt and ko, respectively. Bar graph on the right show the semi-quantitation of the moesin immunofluorescence in slc4a10 wt and ko mouse CPE (wt: n = 5, ko: n = 4). Arrows indicate the luminal membrane, while arrowheads indicate the basolateral membrane. Cell nuclei were visualized by Topro nuclear staining (blue).
Mentions: Previously, we have characterized the slc4a10 ko mouse (Jacobs et al., 2008) and showed that the cellular localization of NHE1 in these mice was found in the basolateral plasma membrane instead of in the luminal membrane as in the slc4a10 wt mouse (Damkier et al., 2009). Therefore, antibodies reacting specifically against the ezrin and moesin proteins known to interact with NHE1 directly or indirectly were used for immunohistochemical analysis of CPE. As previously observed, ezrin staining is found in the cytoplasm close to the luminal plasma membrane in the wt CPE (Figure 4A, micrographs). Ezrin staining was more variable among slc4a10 ko mice, but was also here found predominantly in the subluminal domain in the CPE as assessed by immunofluorescence (Figure 4B). Figure 5 shows that the ezrin distribution appears more cytosolic in slc4a10 ko mice compared to slc4a10 wt in peroxidase stained sections from the same mice. Some labeling is observed in the luminal membrane domain, but the labeling is also found in intracellular compartments. The bar graph in Figure 4A shows that the semi-quantified immunofluorescence signal for ezrin did not differ between slc4a10 wt and ko mice, (p = 0.556, n = 5 and 4 for wt and ko, respectively).

Bottom Line: Anion exchanger 2 abundance is increased in slc4a10 knockout and its anchor protein, α-adducin is almost exclusively found near the basolateral domain.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, Faculty of Health, Aarhus University Aarhus, Denmark.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus