Kim CY et al. (2023), Ruvbl1 is Essential for Ciliary Beating during ...

XB-ART-60459

Dev Reprod 2023 Sep 01;273:159-165. doi: 10.12717/DR.2023.27.3.159.

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Ruvbl1 is Essential for Ciliary Beating during Xenopus laevis Embryogenesis.

Kim CY .

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The Ruvb-like AAA ATPase1 (Ruvbl1; also known as Pontin) is an evolutionary conserved protein belonging to the adenosine triphosphates associated with diverse cellular activities (AAA+) superfamily of ATPases. Ruvbl1 is a component of various protein supercomplexes and is involved in a variety of cellular activities, including chromatin remodeling, DNA damage repair, and mitotic spindle assembly however, the developmental significance of this protein is unknown and needs detailed investigation. We investigated the developmental significance of Ruvbl1 in multiciliated cells of the Xenopus laevis epidermis since ruvbl1 is expressed in the multiciliated cells and pronephros during X. laevis embryogenesis. The knockdown of ruvbl1 significantly impaired cilia-driven fluid flow and basal body polarity in the X. laevis epidermis compared to control embryos, but did not affect cilia morphology. Our results suggest that Ruvbl1 plays a significant role in embryonic development by regulating ciliary beating; however, further investigation is needed to determine the mechanisms involved.

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Genes referenced: ruvbl1

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	Fig. 1. Spatio-temporal expression patterns of ruvbl1 during Xenopus laevis embryogenesis.(A) Temporal expression patterns by RT-PCR. Ruvbl1 is expressed maternally throughout all stages. Ornithine decarboxylase (odc) is used as a loading control. (B) Spatial expression patterns via whole-mount in situ hybridization. Digoxigenin–labeled antisense probes for ruvbl1 were used. At the late neurula stage (St. 17 and 20), ruvbl1 is expressed as dots in the epidermis. At the tailbud stage, ruvbl1 is expressed in the epidermis, branchial arches, eyes, and brain. At stage 35, ruvbl1 is expressed in the pronephros and nephrostome (scale bar=250 μm). (C) Double staining with WISH of ruvbl1 (purple) and immunofluorescence for multicilaited cells using acetylated tubulin (red) on the same embryos (scale bar=250 μm). ba, branchial arch; pn, pronephros; ns, nephrostomes.
	Fig. 2. Knockdown of ruvbl1 perturbed ciliary movement in Xenopus laevis epidermis.(A) Control MO (32 ng) or ruvbl1 MO (32 ng) was injected into one–cell staged embryos. Injected embryos were collected at stage 28 and treated with fluorescent microbeads on their head side. The microbead flow from head to tail was recorded using the Tracker 6.0 program. (B) Bead velocity graph. A total of 32 beads from 8 embryos for each control MO or ruvbl1 MO-injected embryo. Ruvbl1 morphants had significantly reduced microbead flow. * p<0.05; p<0.01; * p<0.001; **** p<0.0001.
	Fig. 3. Knockdown of Ruvbl1 led to defective basal body polarity in Xenopus laevis embryos.(A) Embryos were injected with control MO or ruvbl1 MO and fixed in MEMFA at stage 32. Motile cilia were stained green with acetylated tubulin and observed via confocal microscopy. The depletion of ruvbl1 did not affect the number of cilia in multiciliated cells (scale bar=20 μm). (B) Embryos were injected with centrin-RFP and clamp-GFP with each MO to visualize basal body polarity, which is illustrated by white arrows. Ruvbl1 morphants had defective basal body polarity compared to the control MO-injected embryos.

References [+] :

Bereshchenko, Pontin is essential for murine hematopoietic stem cell survival. 2012, Pubmed