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The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea.

Kirjavainen A, Laos M, Anttonen T, Pirvola U - Biol Open (2015)

Bottom Line: Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion.Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network.In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Viikinkaari 1, 00014 University of Helsinki, Finland.

No MeSH data available.


Altered aPKCζ expression in outer hair cells of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice.Confocal images of whole mount specimens dissected from the medial cochlear coil of control and mutant mice at E18.5 and labeled for F-actin, acetylated tubulin and aPKCζ. All images are shown in medial-lateral orientation, as indicated in (A). (A–A″) In the control specimen, aPKC is expressed in the cortical and, weaker, in the cytoplasmic domain at the OHC surface, medial to the hair bundle. Asterisks mark the lateral, aPKC-negative domain. (B–B″) In the mutant specimen, both cortical and cytoplasmic aPKC expression domains are rotated with respect to hair bundle misorientation. (C–C′″) In the mutant specimen, triple-labeling for acetylated tubulin, phalloidin and aPKC shows that the rotation of aPKC expression parallels the rotation of the astral microtubule network and hair bundle misorientation. (D–D″) High magnification views of two OHCs with abnormal bundles. The OHC below has a completely turned bundle. Asterisks mark the concise aPKC-free area in these cells (compare to control OHCs in Fig. 8A′). Scale bar shown in D″: A–C′″, 5 µm; D–D″, 2 µm.
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f08: Altered aPKCζ expression in outer hair cells of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice.Confocal images of whole mount specimens dissected from the medial cochlear coil of control and mutant mice at E18.5 and labeled for F-actin, acetylated tubulin and aPKCζ. All images are shown in medial-lateral orientation, as indicated in (A). (A–A″) In the control specimen, aPKC is expressed in the cortical and, weaker, in the cytoplasmic domain at the OHC surface, medial to the hair bundle. Asterisks mark the lateral, aPKC-negative domain. (B–B″) In the mutant specimen, both cortical and cytoplasmic aPKC expression domains are rotated with respect to hair bundle misorientation. (C–C′″) In the mutant specimen, triple-labeling for acetylated tubulin, phalloidin and aPKC shows that the rotation of aPKC expression parallels the rotation of the astral microtubule network and hair bundle misorientation. (D–D″) High magnification views of two OHCs with abnormal bundles. The OHC below has a completely turned bundle. Asterisks mark the concise aPKC-free area in these cells (compare to control OHCs in Fig. 8A′). Scale bar shown in D″: A–C′″, 5 µm; D–D″, 2 µm.

Mentions: In several cell types, Cdc42 regulates polarization through aPKC, and this signaling has been shown to control cell polarity through the regulation of microtubule dynamics (Etienne-Manneville, 2004; Etienne-Manneville et al., 2005). Therefore, we next focused on aPKC expression at the OHC's apical surface. It has been previously shown that the aPKC isoform λ/ι is not expressed in the OC around birth (P2), but becomes upregulated a few days thereafter (Anttonen et al., 2012). We found that the aPKCζ isoform is expressed in the hair cell apex during late-embryogenesis. Most prominent expression was found in the medial plasma membrane, opposite to the vertex of the hair bundle (Fig. 8A–A″). Weaker cytoplasmic expression was seen on the medial side at the cell surface (Fig. 8A–A″). These findings are consistent with prior data revealing that the hair bundle is positioned at the interface of aPKC-positive and -negative domains at the hair cell apex (Ezan et al., 2013; Tarchini et al., 2013). Based on triple-labeling with phalloidin and antibodies against ZO-1 and aPKCζ or phospho-aPKC (marking activated kinase), aPKCζ (data not shown) and phospho-aPKC (supplementary material Fig. S3) were localized to the level of tight junctions of OHCs.


The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea.

Kirjavainen A, Laos M, Anttonen T, Pirvola U - Biol Open (2015)

Altered aPKCζ expression in outer hair cells of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice.Confocal images of whole mount specimens dissected from the medial cochlear coil of control and mutant mice at E18.5 and labeled for F-actin, acetylated tubulin and aPKCζ. All images are shown in medial-lateral orientation, as indicated in (A). (A–A″) In the control specimen, aPKC is expressed in the cortical and, weaker, in the cytoplasmic domain at the OHC surface, medial to the hair bundle. Asterisks mark the lateral, aPKC-negative domain. (B–B″) In the mutant specimen, both cortical and cytoplasmic aPKC expression domains are rotated with respect to hair bundle misorientation. (C–C′″) In the mutant specimen, triple-labeling for acetylated tubulin, phalloidin and aPKC shows that the rotation of aPKC expression parallels the rotation of the astral microtubule network and hair bundle misorientation. (D–D″) High magnification views of two OHCs with abnormal bundles. The OHC below has a completely turned bundle. Asterisks mark the concise aPKC-free area in these cells (compare to control OHCs in Fig. 8A′). Scale bar shown in D″: A–C′″, 5 µm; D–D″, 2 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400594&req=5

f08: Altered aPKCζ expression in outer hair cells of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice.Confocal images of whole mount specimens dissected from the medial cochlear coil of control and mutant mice at E18.5 and labeled for F-actin, acetylated tubulin and aPKCζ. All images are shown in medial-lateral orientation, as indicated in (A). (A–A″) In the control specimen, aPKC is expressed in the cortical and, weaker, in the cytoplasmic domain at the OHC surface, medial to the hair bundle. Asterisks mark the lateral, aPKC-negative domain. (B–B″) In the mutant specimen, both cortical and cytoplasmic aPKC expression domains are rotated with respect to hair bundle misorientation. (C–C′″) In the mutant specimen, triple-labeling for acetylated tubulin, phalloidin and aPKC shows that the rotation of aPKC expression parallels the rotation of the astral microtubule network and hair bundle misorientation. (D–D″) High magnification views of two OHCs with abnormal bundles. The OHC below has a completely turned bundle. Asterisks mark the concise aPKC-free area in these cells (compare to control OHCs in Fig. 8A′). Scale bar shown in D″: A–C′″, 5 µm; D–D″, 2 µm.
Mentions: In several cell types, Cdc42 regulates polarization through aPKC, and this signaling has been shown to control cell polarity through the regulation of microtubule dynamics (Etienne-Manneville, 2004; Etienne-Manneville et al., 2005). Therefore, we next focused on aPKC expression at the OHC's apical surface. It has been previously shown that the aPKC isoform λ/ι is not expressed in the OC around birth (P2), but becomes upregulated a few days thereafter (Anttonen et al., 2012). We found that the aPKCζ isoform is expressed in the hair cell apex during late-embryogenesis. Most prominent expression was found in the medial plasma membrane, opposite to the vertex of the hair bundle (Fig. 8A–A″). Weaker cytoplasmic expression was seen on the medial side at the cell surface (Fig. 8A–A″). These findings are consistent with prior data revealing that the hair bundle is positioned at the interface of aPKC-positive and -negative domains at the hair cell apex (Ezan et al., 2013; Tarchini et al., 2013). Based on triple-labeling with phalloidin and antibodies against ZO-1 and aPKCζ or phospho-aPKC (marking activated kinase), aPKCζ (data not shown) and phospho-aPKC (supplementary material Fig. S3) were localized to the level of tight junctions of OHCs.

Bottom Line: Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion.Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network.In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Viikinkaari 1, 00014 University of Helsinki, Finland.

No MeSH data available.