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.
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DOI：
10.1016/j.bbrc.2021.02.091
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发表时间：
2021-03
影响因子：
3.1
通讯作者：
A. Tsutiya;M. Arito;Takuma Tagashira;Masaaki Sato;K. Omoteyama;Toshiyuki Sato;N. Suematsu;M. Kurokawa;Tomohiro Kato
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文献类型：
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作者：
A. Tsutiya;M. Arito;Takuma Tagashira;Masaaki Sato;K. Omoteyama;Toshiyuki Sato;N. Suematsu;M. Kurokawa;Tomohiro Kato
目的layilin是一种具有C型凝集素基序的1型跨膜蛋白，其功能尚不清楚。在这里，我们研究了精确的细胞内定位layilin和位置相关的functions.MethodsWe使用HEK293 T细胞，以评估layilin与不同的个人细胞器标记的双重免疫染色的共定位。然后，我们研究了在layilin-knockdown（KD）条件下的线粒体形态，也与免疫染色。接下来，我们通过蛋白质印迹法测量了layilin-KD细胞与对照细胞中参与线粒体动力学调节的蛋白质，DRP 1，pS616-DRP 1，mitofusin 1，mitofusin 2，CDK 1，pY15-CDK 1和细胞周期蛋白B1的量。此外，通过使用layilin-knockout（KO）细胞，CDK1和pY15-CDK1的量以及线粒体形态学进行了研究。在对照细胞中观察到小圆形线粒体，而在layilin-KD细胞中观察到细长且高度连接的线粒体。在layilin-KD细胞中，活性DRP1（pS616-DRP1）和总DRP1的量显著小于对照。Layilin-KD细胞中失活的CDK1（pY15-CDK1）的量显著大于对照。在layilin-KD细胞中没有其他测试的分子显著改变。在layilin-KO细胞中失活的CDK1的量显著大于野生型（WT）细胞。在WT细胞中观察到小的圆形线粒体，而在layilin-KO cells. ConclusionWe在这里证明layilin发挥了作用，在维护破碎的线粒体在线粒体动力学，这一功能需要CDK1和DRP1激活的线粒体中观察到细长和高度连接。我们的数据揭示了layilin的新功能，即调节线粒体动力学。
ObjectFunctions 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.MethodsWe 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.ResultWe 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.ConclusionWe 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.