Tu F et al. (2018), Protein localization screening in vivo reveals ...

XB-ART-56194

J Cell Sci 2018 Jan 29;1313:. doi: 10.1242/jcs.206565.

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Protein localization screening in vivo reveals novel regulators of multiciliated cell development and function.

Tu F , Sedzinski J , Ma Y , Marcotte EM , Wallingford JB .

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Multiciliated cells (MCCs) drive fluid flow in diverse tubular organs and are essential for the development and homeostasis of the vertebrate central nervous system, airway and reproductive tracts. These cells are characterized by dozens or hundreds of motile cilia that beat in a coordinated and polarized manner. In recent years, genomic studies have not only elucidated the transcriptional hierarchy for MCC specification but also identified myriad new proteins that govern MCC ciliogenesis, cilia beating and cilia polarization. Interestingly, this burst of genomic data has also highlighted that proteins with no obvious role in cilia do, in fact, have important ciliary functions. Understanding the function of proteins with little prior history of study presents a special challenge, especially when faced with large numbers of such proteins. Here, we define the subcellular localization in MCCs of ∼200 proteins not previously implicated in cilia biology. Functional analyses arising from the screen provide novel links between actin cytoskeleton and MCC ciliogenesis.

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Species referenced: Xenopus laevis
Genes referenced: arfgap3 c7h10orf88 ccp110 cep164 cetn4 dennd2b eef1a2 efhc2 galt ift20 mcc ptk2 rfx2 ttbk2
GO keywords: cytosolic ciliogenesis

???displayArticle.omims??? HYDROCEPHALUS, NORMAL-PRESSURE, 1; HYDNP1

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	Fig. 1. High-content screening of protein localization in MCCs in vivo. (A) Schematics of the screening pipeline; see Materials and Methods for details. (B) Schematics of MCC subcellular structures, indicating the major distinct subcellular structures identified in our screen. (C) Summary of screening results. Out of 259 candidates, 198 showed detectable signal localized to distinct subcellular structures, as categorized on the histogram. (D) Representative localizations of screened Rfx2 targets. Left columns, expression patterns of selected genes; middle columns, expression patterns of reference genes. Afap1 localizes to the actin cortex (marked by LifeAct–RFP), Efhc2 to axonemes (marked by CAAX–RFP), Ablim1 to basal bodies (marked by Centrin2–BFP), Dap3 to mitochondria (marked by mito-RFP), Tmem38b to ER (marked by Cal–BFP-KDEL), Arfgap3 to the Golgi (marked by GalT–RFP), C10orf88 to cytosol, and Fam125b to the basolateral membrane (marked by CAAX–RFP). Numbers at the bottom-left corner indicate the z-plane position in reference to the apical domain (0 µm z). Scale bars: 5 μm. The yellow dotted line outlines the cell boundary of MCC.
	Fig. 2. Myo5c localizes to basal bodies and is required for basal body apical migration. (A) Myo5c–GFP (green) localized in proximity to basal bodies (marked by Centrin2–RFP, magenta) and aligned with actin cables (as marked by LifeAct–BFP, cyan). Images were taken at stage 19 and at 3 µm below (−3 μm Z) the apical surface of the MCC (outlined by a yellow dotted line). The orange square highlighted the region shown at a higher magnification in the four right-most panels. Scale bar: 1 µm. (B) Sagittal sections of early stage intercalating MCCs showing basal bodies (marked by Chibby–GFP, green) apparently migrating along actin cables (marked by phalloidin, magenta) to the apical surface of MCC. G, goblet cell. Scale bars: 5 µm. (C) The dominant negative (DN) form of Myo5c was generated by truncating the myosin motor domain of Myo5c (amino acids 84–744) to disrupt its migration ability, while leaving the cargo-binding domain (amino acids 1346–1713) intact. (D) Overexpression of the dominant-negative version of Myo5c (Myo5c-DN–GFP driven by α-tubulin promoter) disrupted basal body migration. In controls (GFP driven by α-tubulin promoter), most of the basal bodies (white) docked within the apical actin network (marked by phalloidin, magenta, and outlined by a yellow dotted line). Upon overexpression of Myo5c-DN, basal bodies failed to migrate apically and accumulated below the apical surface, see orthogonal views. Scale bars: 10 µm. (E) Quantification (mean±s.d.) of basal body positions in controls and upon overexpression of a Myo5c-DN in MCCs. More than 75% of the basal bodies in Myo5c-DN-overexpressing cells failed to migrate to the apical surface of MCCs. The mean±s.d. depth of basal bodies increased from 0.08±0.06 µm in controls to 1.37±0.35 µm below the apical domain (reference position, 0 µm) in Myo5-DN-expressing cells (***P<0.001, Mann–Whitney U-test; control, n=13 cells; Myo5C-DN, n=14 cells; N>5 embryos).
	Fig. 3. St5 localizes to apical actin networks and is required for basal body planar polarity. (A) GFP–St5 (green) localized to the apical and subapical actin network (marked by phalloidin, magenta). Scale bars: 10 µm (main images); 1 µm (magnified image). Images were taken at stage 32. (B) St5 knockdown (KD) reduced the number of subapical actin foci (actin marked by phalloidin, magenta). Scale bars: 10 µm. (C) The number of subapical actin foci was decreased from 61.3±22.7 in controls to 8.7±9.7 in St5 KD (P<0.001, Mann–Whitney U-test; control, n=21 cells; St5 KD, n=33 cells; N>5 embryos). Data represent mean±s.d. (D) St5 KD disrupts basal body orientation. Orientation of basal bodies was determined by measuring the angle (yellow dotted line) between a basal body (marked by Centrin2, white) and its corresponding rootlet (marked by Clamp, green) in respect to the horizontal line. Scale bar: 10 µm (top panel), 1 µm (bottom panel). (E) Quantification of basal bodies orientation. Each arrow represents one cell, where length indicates uniformity of measured angle (as shown in D) in that cell (resultant vector). The mean±s.d. resultant vector value was decreased from 0.71±0.18 in controls to 0.39±0.2 in the St5 KD cells. (P<0.001, Mann–Whitney U-test; control, n=19 cells; St5 KD, n=22 cells; N>5 embryos).
	Fig. 4. St5 is required for ciliogenesis. (A) Perturbation of St5 disrupted ciliogenesis. Left panel, axonemes visualized by CAAX–RFP, magenta. Right panel, SEM of a control MCC and MCC upon St5 knockdown (KD). Scale bars: 10 µm. (B) The axoneme number per MCC was significantly reduced from 67.1±15.4 in controls to 14.0±8.2 in St5 KD (**P<0.001, Mann–Whitney U-test; Control, n=14 cells; St5 KD, n=32 cells; N>5 embryos). The number of axonemes was increased by rescue expression of GFP–St5 with an α-tubulin promoter to 18.6±8.5 (P<0.05, Mann–Whitney U-test; St5 KD with GFP–St5, n=25 cells, N>5 embryos). Data represent mean±s.d. (C) St5 KD did not disrupt the recruitment of Ift20 (Ift–GFP, green). Scale bars: 10 µm. (D) St5 KD increases the level of GFP–Cp110 (green) at basal bodies (marked by Centrin2–RFP, white). Scale bars: 10 µm. (E) The normalized Cp110 intensities around basal bodies increased from 0.046±0.045 to 0.093±0.098. (*P<0.001, Mann–Whitney U-test; Control, n=1430 intensities from eight cells; St5 KD, n=1413 from 10 cells; N>5 embryos). Data represent mean±s.d. (F) St5 is dispensable for Ttbk2 recruitment. GFP–Ttbk2 showed ring shape structures in both control and St5 KD cells. (G) Network of St5-containing protein complex predicted by assembled map of human protein complexes (Drew et al., 2017). Line weights represent support vector machine confidence scores. (H) The level of Cep162–GFP (green) at basal bodies (marked by Centrin2–RFP, white) was decreased upon St5 KD. Scale bars: 10 µm. (I) The normalized Cep162 intensities around basal bodies decreased from 0.28±0.21 to 0.07±0.10. (*P<0.001, Mann–Whitney U-test; Control, n=1734 intensities from 10 cells; St5 KD, n=1710 from 13 cells; N>5 embryos). Data represent mean±s.d. The yellow dotted lines in D and H outline the cell boundary of MCCs.
	Figure S1. Subcellular localization of selected proteins from the screen in MCCs. (A) Arfgap3-GFP (green) localized to Golgi (Golgi marker, GalT-RFP, red), 3D view. (B) GFP-Pof1b(green) localized to the junction of MCC (membrane marker, CAAX-RFP, magenta), 3D view. (C) Fam125b-GFP (green) localized to basolateral membrane (CAAX-RFP, magenta), 3D view. (D) Image sequences of a MCC expressing CCDC146-GFP(green). CCDC146 localized to cytoplasmic foci of different sizes. White arrows indicate formation of a new focus. Yellow dotted line outlines the cell boundary of MCC. Scale bar, 10 μm. (E) An MCC from an embryo injected with Ccdc167GFP plasmid, Adarb1RFP plasmid and memBFP mRNA together.
	Figure S2. St5 KD and KO show similar axonemogenesis defect phenotype but St5 is not required for recruitment of Cep164 and Fak. (A) The levels of St5 transcripts were significantly reduced by injection of St5 morpholino compared with control. EF1a, input reference. (B) T7 Endonuclease I (T7EI) assay showed St5 CRISPR induced genomic mutations in St5 (C) St5 MO#2 and St5 CRISPR led to similar axonemogenesis defects as St5 MO#1. (D) St5 KD did not disrupt basal body docking. (E) St5 KD did not disrupt the recruitment of Fak (Fak-GFP, green). Scale bar, 10 μm. (F) St5 KD did not disrupt the transition zone marked by Cep164(GFP-Cep164, green). Scale bar, 10 μm.

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