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KIF3A regulates the Wnt/β-catenin pathway via transporting β-catenin during spermatogenesis in Eriocheir sinensis

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Abstract

The Wnt/β-catenin pathway participates in many important physiological events such as cell proliferation and differentiation in the male reproductive system. We found that Kinesin-2 motor KIF3A is highly expressed during spermatogenesis in Eriocheir sinensis; it may potentially promote the intracellular transport of cargoes in this process. However, only a few studies have focused on the relationship between KIF3A and the Wnt/β-catenin pathway in the male reproductive system of decapod crustaceans. In this study, we cloned and characterized the CDS of β-catenin in E. sinensis for the first time. Fluorescence in situ hybridization and immunofluorescence results showed the colocalization of Es-KIF3A and Es-β-catenin at the mRNA and the protein level respectively. To further explore the regulatory function of Es-KIF3A to the Wnt/β-catenin pathway, the es-kif3a was knocked down by double-stranded RNA (dsRNA) in vivo and in primary cultured cells in testes of E. sinensis. Results showed that the expression of es-β-catenin and es-dvl were decreased in the es-kif3a knockdown group. The protein expression level of Es-β-catenin was also reduced and the location of Es-β-catenin was changed from nucleus to cytoplasm in the late stage of spermatogenesis when es-kif3a was knocked down. Besides, the co-IP result demonstrated that Es-KIF3A could bind with Es-β-catenin. In summary, this study indicates that Es-KIF3A can positively regulate the Wnt/β-catenin pathway during spermatogenesis and Es-KIF3A can bind with Es-β-catenin to facilitate the nuclear translocation of Es-β-catenin.

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Funding

This project was supported by the National Natural Science Foundation of China (Nos. 41776144 and 31572603) and the Natural Science Foundation of Zhejiang Province, China (No. LY20H040010).

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Authors and Affiliations

  1. The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China

    Ya-Jing Liang, Jia-Hao Ni, Lan-Min Wang & Wan-Xi Yang

  2. The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, China

    Fu-Qing Tan

Authors
  1. Ya-Jing Liang
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  2. Jia-Hao Ni
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  3. Lan-Min Wang
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  4. Fu-Qing Tan
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Correspondence to Wan-Xi Yang.

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The authors declare that they have no conflict of interest.

Ethical statement

The animal use in this study was approved by Animal Experimental Ethical Inspection of the First Affiliated Hospital, College of Medicine, Zhejiang University (Reference Number: 2019-1061).

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Fig. S1

The CDS sequence of Es-β-catenin. The start codon is “ATG” in green, and “TAG” in red is the stop codon. The amino acid sequence which is encoded by DNA is in gray. The 2451 bp open reading frame encodes 816 amino acids. (PNG 1822 kb) (PNG 3163 kb)

High Resolution (TIF 4853 kb)

Fig. S2

The major structural features of Es-β-catenin. (a) The secondary structure predicted by SMART shows that there is a coiled coil region and two ARM functional domains. (b) The globular three-dimensional structure was predicted by NCBI. (PNG 1822 kb)

High Resolution (TIF 1102 kb)

Fig. S3

The multiple sequence alignment of Es-β-catenin. The amino acid sequence of β-catenin in E.sinensis and other species was aligned by Vector NTI11.5 and DNAMAN8.0. The sequence in pink means the homology is 100%, The sequence in blue means the homology is >70%. A conserved functional domain had been found in Es-β-catenin in red box. (PNG 3857 kb)

High Resolution (TIF 1713 kb)

Fig. S4

The phylogenetic tree analysis of Es-β-catenin. This tree shows the phylogenetic analysis of Es-β-catenin and its homologs in other species which were constructed by Neighbor-Joining (NJ) in Mega 7.0 software. The dark triangle marked the status of Es-β-catenin. (PNG 7057 kb)

High Resolution (TIF 9727 kb)

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Liang, YJ., Ni, JH., Wang, LM. et al. KIF3A regulates the Wnt/β-catenin pathway via transporting β-catenin during spermatogenesis in Eriocheir sinensis. Cell Tissue Res 381, 527–541 (2020). https://doi.org/10.1007/s00441-020-03220-x

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