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Flattop regulates basal body docking and positioning in mono- and multiciliated cells.

Gegg M, Böttcher A, Burtscher I, Hasenoeder S, Van Campenhout C, Aichler M, Walch A, Grant SG, Lickert H - Elife (2014)

Bottom Line: Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells.In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3, and Fltp localize directly adjacent to the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton.Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease, and asymmetric cell division.

View Article: PubMed Central - PubMed

Affiliation: Institute of Stem Cell Research, Helmholtz Center Munich, Munich, Germany.

ABSTRACT
Planar cell polarity (PCP) regulates basal body (BB) docking and positioning during cilia formation, but the underlying mechanisms remain elusive. In this study, we investigate the uncharacterized gene Flattop (Fltp) that is transcriptionally activated during PCP acquisition in ciliated tissues. Fltp knock-out mice show BB docking and ciliogenesis defects in multiciliated lung cells. Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells. In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3, and Fltp localize directly adjacent to the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton. Dlg3 and Fltp knock-outs suggest that both cooperatively translate PCP cues for BB positioning in the inner ear. Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease, and asymmetric cell division.

No MeSH data available.


Related in: MedlinePlus

ALI differentiation efficiency and quantification of BB docking defect.(A) Graph showing the percentage of cells in stage II–IV for WT and FltpZV/ZV (HOM) ALIs at day zero (before the switch to ALI condition), one, two, and four measured by GFP and pericentrin (HOM) and only pericentrin (WT) staining compared to the total amount of cells in the ALI culture stained by DAPI (representative images were analyzed). For WT analysis we counted n = 1 (1493 cells) for day 0, n = 1 (1345) for day 1, n = 2 (1635) for day 2, n = 2 (1262) for day 4 and for HOM n = 1 (1367 cells) for day 0, n = 1 (1542) for day 1, n = 3 (942) for day 2, n = 4 (1123) for day 4. (B) Shows the percentage of ciliation (measured by acetylated-Tubulin staining) of cells with amplified centrosomes (measured by pericentrin staining) for WT and HOM ALIs at day two and four categorized into stage II–III and stage IV. For WT analysis we counted n = 4 (609 cells) for day 2, n = 4 (231) for day 4 and for HOM n = 10 (611) for day 2, n = 7 (286) for day 4.DOI:http://dx.doi.org/10.7554/eLife.03842.011
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fig4s1: ALI differentiation efficiency and quantification of BB docking defect.(A) Graph showing the percentage of cells in stage II–IV for WT and FltpZV/ZV (HOM) ALIs at day zero (before the switch to ALI condition), one, two, and four measured by GFP and pericentrin (HOM) and only pericentrin (WT) staining compared to the total amount of cells in the ALI culture stained by DAPI (representative images were analyzed). For WT analysis we counted n = 1 (1493 cells) for day 0, n = 1 (1345) for day 1, n = 2 (1635) for day 2, n = 2 (1262) for day 4 and for HOM n = 1 (1367 cells) for day 0, n = 1 (1542) for day 1, n = 3 (942) for day 2, n = 4 (1123) for day 4. (B) Shows the percentage of ciliation (measured by acetylated-Tubulin staining) of cells with amplified centrosomes (measured by pericentrin staining) for WT and HOM ALIs at day two and four categorized into stage II–III and stage IV. For WT analysis we counted n = 4 (609 cells) for day 2, n = 4 (231) for day 4 and for HOM n = 10 (611) for day 2, n = 7 (286) for day 4.DOI:http://dx.doi.org/10.7554/eLife.03842.011

Mentions: To analyze the dynamic cellular and molecular requirement for Fltp during BB docking, cilia formation, and PCP establishment, we employed the mTEC culture system (You et al., 2002; Vladar et al., 2012). Switching mTEC culture to ALI conditions induces a differentiation process of lung progenitor cells towards a MCC phenotype. During differentiation the progenitor cells pass through different maturation stages (Figure 4A): Monociliated lung progenitor cells (stage I) start centrosome/BB amplification followed by BB transport and docking (stage II–III) and subsequent cilia formation (stage IV) (Vladar et al., 2012). In these cultures, Fltp-driven H2B-Venus reporter activity is activated at the transition from stage I to stage II when centrosomes/BBs are amplified and docked in heterozygous and homozygous Fltp ALI cultures (Figure 4B). Fltp-H2B-Venus expression increases during maturation until cells are terminally differentiated. This correlates with active PCP signaling and initial asymmetric core component localization (Vladar et al., 2012). Subcellular co-localization studies further revealed that Fltp is localized to the apical, but not sub-apical actin cytoskeleton and fills the gaps in the actin network from where MT-based cilia project (Figure 4C–Cʹʹʹ,E). Moreover, Fltp co-localizes with newly synthesized MT plus ends that are labeled with anti-EB1 antibodies (Figure 4D). Together with the direct adjacent localization of Fltp next to the pericentriolar matrix stained by γ-Tubulin (Figure 4E), these data suggest that Fltp connects BBs and ciliary MT plus ends to the cortical actin cytoskeleton. To test this idea, we examined mTECs switched to ALI conditions from Fltp+/+ and FltpZV/ZV mice at day two and four of differentiation qualitatively and quantitatively. Fltp+/+ and FltpZV/ZV differentiated with comparable efficiencies and speed as measured by Fltp-driven H2B-GFP expression and centrosome amplification (Figure 4—figure supplement 1A), and a comparable number of centrosomes are formed in differentiating Fltp+/+ and FltpZV/ZV mTECs (Figure 4F,G), whereas BB docking and cilia formation was delayed at ALI+2 and +4 days of differentiation (Figure 4F–I, Figure 4—figure supplement 1B). Taken together, these data suggest that Fltp functions in the process of BB docking and cilia formation.10.7554/eLife.03842.010Figure 4.Fltp is expressed and necessary during BB docking.


Flattop regulates basal body docking and positioning in mono- and multiciliated cells.

Gegg M, Böttcher A, Burtscher I, Hasenoeder S, Van Campenhout C, Aichler M, Walch A, Grant SG, Lickert H - Elife (2014)

ALI differentiation efficiency and quantification of BB docking defect.(A) Graph showing the percentage of cells in stage II–IV for WT and FltpZV/ZV (HOM) ALIs at day zero (before the switch to ALI condition), one, two, and four measured by GFP and pericentrin (HOM) and only pericentrin (WT) staining compared to the total amount of cells in the ALI culture stained by DAPI (representative images were analyzed). For WT analysis we counted n = 1 (1493 cells) for day 0, n = 1 (1345) for day 1, n = 2 (1635) for day 2, n = 2 (1262) for day 4 and for HOM n = 1 (1367 cells) for day 0, n = 1 (1542) for day 1, n = 3 (942) for day 2, n = 4 (1123) for day 4. (B) Shows the percentage of ciliation (measured by acetylated-Tubulin staining) of cells with amplified centrosomes (measured by pericentrin staining) for WT and HOM ALIs at day two and four categorized into stage II–III and stage IV. For WT analysis we counted n = 4 (609 cells) for day 2, n = 4 (231) for day 4 and for HOM n = 10 (611) for day 2, n = 7 (286) for day 4.DOI:http://dx.doi.org/10.7554/eLife.03842.011
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221739&req=5

fig4s1: ALI differentiation efficiency and quantification of BB docking defect.(A) Graph showing the percentage of cells in stage II–IV for WT and FltpZV/ZV (HOM) ALIs at day zero (before the switch to ALI condition), one, two, and four measured by GFP and pericentrin (HOM) and only pericentrin (WT) staining compared to the total amount of cells in the ALI culture stained by DAPI (representative images were analyzed). For WT analysis we counted n = 1 (1493 cells) for day 0, n = 1 (1345) for day 1, n = 2 (1635) for day 2, n = 2 (1262) for day 4 and for HOM n = 1 (1367 cells) for day 0, n = 1 (1542) for day 1, n = 3 (942) for day 2, n = 4 (1123) for day 4. (B) Shows the percentage of ciliation (measured by acetylated-Tubulin staining) of cells with amplified centrosomes (measured by pericentrin staining) for WT and HOM ALIs at day two and four categorized into stage II–III and stage IV. For WT analysis we counted n = 4 (609 cells) for day 2, n = 4 (231) for day 4 and for HOM n = 10 (611) for day 2, n = 7 (286) for day 4.DOI:http://dx.doi.org/10.7554/eLife.03842.011
Mentions: To analyze the dynamic cellular and molecular requirement for Fltp during BB docking, cilia formation, and PCP establishment, we employed the mTEC culture system (You et al., 2002; Vladar et al., 2012). Switching mTEC culture to ALI conditions induces a differentiation process of lung progenitor cells towards a MCC phenotype. During differentiation the progenitor cells pass through different maturation stages (Figure 4A): Monociliated lung progenitor cells (stage I) start centrosome/BB amplification followed by BB transport and docking (stage II–III) and subsequent cilia formation (stage IV) (Vladar et al., 2012). In these cultures, Fltp-driven H2B-Venus reporter activity is activated at the transition from stage I to stage II when centrosomes/BBs are amplified and docked in heterozygous and homozygous Fltp ALI cultures (Figure 4B). Fltp-H2B-Venus expression increases during maturation until cells are terminally differentiated. This correlates with active PCP signaling and initial asymmetric core component localization (Vladar et al., 2012). Subcellular co-localization studies further revealed that Fltp is localized to the apical, but not sub-apical actin cytoskeleton and fills the gaps in the actin network from where MT-based cilia project (Figure 4C–Cʹʹʹ,E). Moreover, Fltp co-localizes with newly synthesized MT plus ends that are labeled with anti-EB1 antibodies (Figure 4D). Together with the direct adjacent localization of Fltp next to the pericentriolar matrix stained by γ-Tubulin (Figure 4E), these data suggest that Fltp connects BBs and ciliary MT plus ends to the cortical actin cytoskeleton. To test this idea, we examined mTECs switched to ALI conditions from Fltp+/+ and FltpZV/ZV mice at day two and four of differentiation qualitatively and quantitatively. Fltp+/+ and FltpZV/ZV differentiated with comparable efficiencies and speed as measured by Fltp-driven H2B-GFP expression and centrosome amplification (Figure 4—figure supplement 1A), and a comparable number of centrosomes are formed in differentiating Fltp+/+ and FltpZV/ZV mTECs (Figure 4F,G), whereas BB docking and cilia formation was delayed at ALI+2 and +4 days of differentiation (Figure 4F–I, Figure 4—figure supplement 1B). Taken together, these data suggest that Fltp functions in the process of BB docking and cilia formation.10.7554/eLife.03842.010Figure 4.Fltp is expressed and necessary during BB docking.

Bottom Line: Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells.In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3, and Fltp localize directly adjacent to the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton.Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease, and asymmetric cell division.

View Article: PubMed Central - PubMed

Affiliation: Institute of Stem Cell Research, Helmholtz Center Munich, Munich, Germany.

ABSTRACT
Planar cell polarity (PCP) regulates basal body (BB) docking and positioning during cilia formation, but the underlying mechanisms remain elusive. In this study, we investigate the uncharacterized gene Flattop (Fltp) that is transcriptionally activated during PCP acquisition in ciliated tissues. Fltp knock-out mice show BB docking and ciliogenesis defects in multiciliated lung cells. Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells. In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3, and Fltp localize directly adjacent to the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton. Dlg3 and Fltp knock-outs suggest that both cooperatively translate PCP cues for BB positioning in the inner ear. Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease, and asymmetric cell division.

No MeSH data available.


Related in: MedlinePlus