Reis AH et al. (2021), Identification of the centrosomal maturation fa...

XB-ART-58590

PLoS One 2021 Jan 01;1610:e0259068. doi: 10.1371/journal.pone.0259068.

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Identification of the centrosomal maturation factor SSX2IP as a Wtip-binding partner by targeted proximity biotinylation.

Reis AH , Xiang B , Ossipova O , Itoh K , Sokol SY .

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Wilms tumor-1-interacting protein (Wtip) is a LIM-domain-containing adaptor that links cell junctions with actomyosin complexes and modulates actomyosin contractility and ciliogenesis in Xenopus embryos. The Wtip C-terminus with three LIM domains associates with the actin-binding protein Shroom3 and modulates Shroom3-induced apical constriction in ectoderm cells. By contrast, the N-terminal domain localizes to apical junctions in the ectoderm and basal bodies in skin multiciliated cells, but its interacting partners remain largely unknown. Targeted proximity biotinylation (TPB) using anti-GFP antibody fused to the biotin ligase BirA identified SSX2IP as a candidate protein that binds GFP-WtipN. SSX2IP, also known as Msd1 or ADIP, is a component of cell junctions, centriolar satellite protein and a targeting factor for ciliary membrane proteins. WtipN physically associated with SSX2IP and the two proteins readily formed mixed aggregates in overexpressing cells. By contrast, we observed only partial colocalization of full length Wtip and SSX2IP, suggesting that Wtip adopts a 'closed' conformation in the cell. Furthermore, the double depletion of Wtip and SSX2IP in early embryos uncovered the functional interaction of the two proteins during neural tube closure. Our results suggest that the association of SSX2IP and Wtip is essential for cell junction remodeling and morphogenetic processes that accompany neurulation. We propose that TPB can be a general approach that is applicable to other GFP-tagged proteins.

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Species referenced: Xenopus laevis
Genes referenced: avd frat1 myc shroom3 smo ssx2ip wtip
GO keywords: neural tube closure [+]
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	Fig 1. Biotinylation of the BirA-WtipN fusion in Xenopus embryos. A, Schematic of Wtip. B, C, Embryos were injected with 200 pg of GFP-WtipN RNA, fixed and imaged at the indicated stages. B, GFP-WtipN junctional staining in stage 11 ectoderm. C, GFP-WtipN is localized to basal bodies of skin multiciliated cells (arrows, stage 25). Scale bar, 20 Î¼m. D, Biotinylation of the BirA-WtipN fusion. Four-cell embryos were injected with myc-BirA RNA (300 pg) or mycBirA-WtipN RNA (700 pg) with or without 0.8 mM biotin. Self-biotinylation of mycBirA and mycBirA-WtipN is revealed only in the presence of biotin. Protein levels were assessed with anti-myc antibody. Asterisks indicate endogenous avidin-binding proteins. E. Biotinylated proteins were pulled down with neutravidin beads. In addition to self-biotinylated mycBirA-Wtip, multiple biotinylated bands are detected in the lysates as well as in pulldown.
	Fig 2. Specificity of BirA-GBP-mediated biotinylation. A, GFP-WtipN but not Flag-WtipN is biotinylated (left arrow) by BirA-GBP in the presence of biotin. MycBirA-WtipN is a positive control for biotinylation (right arrow). B, GFP-Prickle3C protein, but not Flag-Prickle3C, was biotinylated by BirA-GBP. Protein expression levels are assessed with tag-specific antibodies as indicated (A, B).
	Fig 3. Identification of SSX2IP by targeted proximity biotinylation. A, Scheme of targeted proximity biotinylation (TPB) with GFP-WtipN. Early embryos were injected with indicated RNAs (200 pg each) and biotin (1.2 mM). B, Biotinylated proteins were enriched with neutravidin agarose beads and immunoblotted with indicated antibodies. Biotinylated GFP-WtipN (asterisk) but not Flag-WtipN in the presence of BirA-GBP was detected in the pulldown. MycBirA-WtipN (asterisk) was biotinylated in the absence of BirA-GBP. C, Top hits identified by SCAFFOLD after LC-MS/MS analysis in both experimental groups but not in the negative control group.
	Fig 4. The physical association of SSX2IP and Wtip. Four-cell embryos were coinjected with GFP-hSSX2IP RNA and either Flag-WtipN or Flag-Wtip RNA (300 pg each). Flag-GFP RNA (300 pg) was the negative control. Embryos were lysed at stage 11 and lysates were immunoprecipitated with anti-Flag agarose beads and immunoblotted with antibodies specific for Flag and GFP. WtipN coprecipitated with SSX2IP more efficiently than full length Wtip.
	Fig 5. SSX2IP and WtipN co-aggregate in ectoderm cells. A, Experimental scheme. HA-RFP-Wtip or HA-RFP-WtipN RNA (50 pg each) was coinjected with GFP-SSX2IP RNA (50 pg) into four-cell embryos. When the embryos reached stage 10 or 12.5, ectoderm was dissected, fixed, and fluorescent protein localization at the cortex and cytoplasmic puncta was imaged at indicated stages. B, GFP-SSX2IP, stage 10.5. C, GFP-SSX2IP, stage 12.5. D, HA-RFP-Wtip, stage 10.5. E, HA-RFP-WtipN, stage 12.5. F-Fâ€, Mixed cytoplasmic aggregates (arrows) in cells coexpressing GFP-SSX2IP and RFP-WtipN, stage 12.5. F, red channel, Fâ€™ green channel, Fâ€, merged image. G, HA-RFP-Wtip, stage 12.5. H-Hâ€, Partial colocalization of GFP-SSX2IP and HA-RFP-Wtip in cytoplasmic puncta (arrows). Scale bar, 15 Î¼m. Images are representative of 3 independent experiments, with 10â€“15 embryos imaged per group in each experiment.
	Fig 6. SSX2IP and Wtip functionally interact to promote neural tube closure. Embryos were injected bilaterally or unilaterally with 10 ng of Wtip MO (Wmo) or SSX2IP MO (Smo) or 20 pg of human SSX2IP RNA as indicated. Neural groove formation along the anteroposterior axis was assessed at stages 16â€“18. A-F, Neural tube closure defects in SSX2IP morphants. A, Scheme of the experiment. B-D, Dorsal views of representative embryos at stages 16â€“17, anterior is up. B, Control uninjected embryo, C, SSX2IP RNA injection. D, Embryo injected with Smo. E, Embryo injected with Smo and SSX2IP RNA. Human SSX2IP RNA partly rescues neural closure defects. Dashed lines indicate neural fold borders. F, Frequency of neural tube (NT) defects. Data are representative of two experiments. G-L, Functional interaction of SSX2IP and Wtip. G, Scheme of the experiment. H-L, Representative embryos at stages 18â€“19 injected with control MO (CoMO, H), Wmo (I), Smo (J) or both Wmo and Smo (K). Midline, white dashed line, neural fold border, magenta dashed line. Anterior is up. L, Frequency of neural tube (NT) defects in the morphants. Data represent three different experiments. In F and L, total number of embryos per group is shown above each bar. Mild, severe and normal phenotypes have been scored separately, with examples shown in L. https://doi.org/10.1371/journal.pone.0259068.g006
	S1Fig. Colocalization of SSX2IP andWtipNin ectoderm cells.A, Experimental scheme.RFP-WtipNRNA (300 pg) andGFP-XSSX2IPRNA (300 pg)were coinjectedinto four-cell embryos. When the embryos reached stage 12.5, they were fixed, and the ectodermal tissue was imaged. B, GFP-SSX2IP localization in ectoderm cells. SSX2IPcolocalizes with WtipN in cytoplasmic puncta. C, RFPWtipN localization at the junctions and cytoplasmic puncta. D-Dâ€™â€™, Coexpression of both proteins revealed mixed cytoplasmic aggregates (arrow). Scale bar, 15 Î¼m. Data are representative of 3 independent experiments.

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