2. Academic Validation
  3. Layilin promotes mitochondrial fission by cyclin-dependent kinase 1 and dynamin-related protein 1 activation in HEK293T cells

Layilin promotes mitochondrial fission by cyclin-dependent kinase 1 and dynamin-related protein 1 activation in HEK293T cells

  • Biochem Biophys Res Commun. 2021 Apr 16:549:143-149. doi: 10.1016/j.bbrc.2021.02.091.
Atsuhiro Tsutiya 1,  Mitsumi Arito 2,  Takuma Tagashira 3,  Masaaki Sato 4,  Kazuki Omoteyama 5,  Toshiyuki Sato 6,  Naoya Suematsu 7,  Manae S Kurokawa 8,  Tomohiro Kato 9
Affiliations

Affiliations

  • 1 Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address: atsuchi@marianna-u.ac.jp.
  • 2 Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address: m-ari@marianna-u.ac.jp.
  • 3 Department of Molecular Biology, Faculty of Pharmaceutical Science, Yokohama University of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama, 245-0066, Japan. Electronic address: tagasitaku0307@gmail.com.
  • 4 Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address: msato@marianna-u.ac.jp.
  • 5 Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address: omoteyama@marianna-u.ac.jp.
  • 6 Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address: stoshi@marianna-u.ac.jp.
  • 7 Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address: n2sue@marianna-u.ac.jp.
  • 8 Disease Biomarker Analysis and Molecular Regulation, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address: manae@marianna-u.ac.jp.
  • 9 Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address: t3kato@marianna-u.ac.jp.
PMID: 33676182 DOI: 10.1016/j.bbrc.2021.02.091
Abstract

Object: Functions of layilin, a type 1 transmembrane protein with a C-type lectin motif, remain to be clarified. We here investigated precise intracellular localization of layilin and the location-related functions.

Methods: We used HEK293T cells to assess the co-localization of layilin with different individual organelle markers by double immunostaining. We then investigated mitochondrial morphology in layilin-knockdown (KD) conditions, also with immunostaining. Next, we measured amounts of proteins involved in regulation of mitochondrial dynamics, DRP1, pS616-DRP1, mitofusin1, mitofusin2, CDK1, pY15-CDK1, and cyclin B1, in layilin-KD cells versus control cells by Western blot. Furthermore, by using layilin-knockout (KO) cells, amounts of CDK1 and pY15-CDK1 as well as mitochondrial morphology were investigated.

Result: We found that layilin localized to mitochondria rather than the Other organelles. Small round-shape mitochondria were observed in control cells, whereas elongated and highly connected mitochondria were observed in layilin-KD cells. Amounts of active DRP1 (pS616-DRP1) and total DRP1 were significantly smaller in layilin-KD cells than in controls. Amounts of inactive CDK1 (pY15-CDK1) were significantly larger in layilin-KD cells than in controls. No Other tested molecules were significantly altered in layilin-KD cells. Amounts of inactive CDK1 were significantly larger in layilin-KO cells than in wild type (WT) cells. Small round-shape mitochondria were observed in WT cells, whereas elongated and highly connected mitochondria were observed in layilin-KO cells.

Conclusion: We here demonstrated that layilin played a role in the maintenance of fragmented mitochondria in mitochondrial dynamics and that this function needed CDK1 and DRP1 activation. Our data unveiled a novel function for layilin, regulation of mitochondrial dynamics.

Keywords

CDK1; DRP1; Layilin; Mitochondria.

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