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. 2021 Apr 16:549:143-149.
doi: 10.1016/j.bbrc.2021.02.091. Epub 2021 Mar 3.

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

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

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

Atsuhiro Tsutiya et al. Biochem Biophys Res Commun. .

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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Conflict of interest statement

Declaration of competing interest A. Tsutiya reports grants from JSPS KAKENHI (Grant Number 19K15752) during the conduct of the study. T. Kato reports grants from Astellas Pharma Inc., grants from CHUGAI PHARMACEUTICAL CO., LTD., grants from Daiichi Sankyo Company, Limited, grants from Pfizer Inc., grants from Sanofi K.K., and grants from Takeda Pharmaceutical Company Limited outside the submitted work. M. Arito, T. Tagashira, M. Sato, K. Omoteyama, T. Sato, N. Suematsu, and M.S. Kurokawa have nothing to disclose.

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