Optogenetic control of YAP can enhance the rate of wound healing
Optogenetic control of YAP can enhance the rate of wound healing
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YAP的光遗传学控制可以提高伤口愈合率
DOI：
10.1101/2022.11.04.515183
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Toh P
中科院分区：
文献类型：
--
作者：
Toh P
损伤后组织需要再生以恢复功能。然而，这种再生能力在不同器官和物种之间差别很大。例如，在心脏中，一些物种在其一生中保持完全的再生能力，但人类心脏细胞显示出有限的修复损伤的能力。心肌梗塞后，心肌细胞功能受损，心脏泵血能力降低，导致心力衰竭。因此，有必要恢复心肌梗死后受损心脏的功能。我们在细胞培养中研究yes相关蛋白（YAP）在驱动损伤后修复中的作用，YAP是一种在生长中起关键作用的转录共调节因子。方法在三种不同细胞系中表达光遗传YAP （optoYAP）。我们利用荧光成像和下游YAP靶基因的实时荧光定量PCR表征了optoYAP的行为和功能。突变体结构是通过定点诱变产生的。采用创面愈合实验对核定位optoYAP进行功能检测。结果利用optoYAP可以精确控制通路激活，我们发现YAP可以诱导下游参与增殖和迁移的基因的表达。optoYAP可促进H9c2型心肌细胞创面愈合。有趣的是，这不是由增殖的增加驱动的，而是由集体细胞迁移驱动的。随后，我们剖析了YAP中特定的磷酸化位点，以确定加速愈合的分子驱动因素。本研究表明，光遗传YAP在H9c2心肌细胞中起作用，其受控激活可能在一系列条件下促进伤口愈合。
BackgroundTissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury.MethodsWe express optogenetic YAP (optoYAP) in three different cell lines. We characterised the behaviour and function of optoYAP using fluorescence imaging and quantitative real-time PCR of downstream YAP target genes. Mutant constructs were generated using site-directed mutagenesis. Nuclear localised optoYAP was functionally tested using wound healing assay.ResultsUtilising optoYAP, which enables precise control of pathway activation, we show that YAP induces the expression of downstream genes involved in proliferation and migration. optoYAP can increase the speed of wound healing in H9c2 cardiomyoblasts. Interestingly, this is not driven by an increase in proliferation, but by collective cell migration. We subsequently dissect specific phosphorylation sites in YAP to identify the molecular driver of accelerated healing.ConclusionsThis study shows that optogenetic YAP is functional in H9c2 cardiomyoblasts and its controlled activation can potentially enhance wound healing in a range of conditions.
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