Keratin-dependent regulation of mitochondria in keratinocytes and mouse epidermis

线粒体的完整性和功能依赖于正确的定位、与内质网的相互作用以及对其动力学的调节。线粒体定位和动力学的机制是由支架和调控蛋白协调的，这些调控蛋白介导了与细胞骨架的相互作用。除了微管外，中间丝蛋白结蛋白、波形蛋白和简单上皮角蛋白可能通过与线粒体相关蛋白plectin 1b、trichoplein和Pirh2相互作用参与线粒体的定位和形状。关于线粒体在哺乳动物表皮中的分布和调控，人们知之甚少。最近的研究表明，线粒体ROS信号而不是ATP对表皮分化和毛囊形态发生至关重要。尽管角蛋白细胞骨架蛋白由于其同型多样性和上下文依赖的功能被理想地定制来调节角质形成细胞中线粒体的定位和活性，但对角蛋白在调节表皮线粒体中的作用知之甚少。我们最近发现角蛋白亚型是桥粒通过PKCalpha黏附的主要调节因子，也是表皮屏障的基本支架。现在，我们发现线粒体在角质形成细胞中的分布是角蛋白依赖性的。在角蛋白缺乏的角质形成细胞中，发现线粒体磷脂酰乙醇胺和心磷脂以及电子传递链复合体1和4蛋白水平增加，导致氧耗量增加30%。最后，角蛋白缺陷小鼠毛囊形态发生缺陷，也报道了小鼠具有表皮特异性的电子传输链缺失。在这里，我们想要了解角蛋白亚型如何在角质形成细胞和体内调节线粒体的位置、动力学和生理学。为了解决这个问题，我们将1)确定介导角蛋白-线粒体相互作用的蛋白质，并确定它们的相互作用区域。除了上述候选蛋白质外，BioID还将用于识别新的相互作用蛋白质。相互作用将通过共聚焦成像和蛋白质相互作用方法来表征，并通过角蛋白调节的角质形成细胞的得失功能研究来验证。2)我们将分析正常角蛋白和突变角蛋白在线粒体-内质网相互作用和线粒体动力学中的作用，以了解角蛋白在分化和角化疾病中如何控制线粒体的定位和生理。3)我们将探讨上述和/或额外的蛋白质和机制是否与角蛋白缺陷小鼠毛囊形态发生缺陷有关。我们的建议将为角蛋白将线粒体与表皮分化的偶联提供新的见解。此外，我们的数据将促进对线粒体和角蛋白紊乱的机制理解，并可能为恢复线粒体的完整性提供新的方法。

Integrity and functionality of mitochondria depend on correct positioning, interaction with the ER and regulation of their dynamics. The mechanisms underlying mitochondrial localization and dynamics are orchestrated by scaffold and regulatory proteins which mediate the interaction with the cytoskeleton. In addition to microtubules, intermediate filament proteins desmin, vimentin and simple epithelial keratins participate in mitochondrial localization and shape, possibly through interactions with mitochondria-associated proteins plectin 1b, trichoplein and Pirh2. Very little is known about mitochondrial distribution and regulation in the mammalian epidermis. Recent studies showed that mitochondrial ROS signalling but not ATP are vital for epidermal differentiation and hair follicle morphogenesis. Although keratin cytoskeletal proteins, due to their isotype diversity and context-dependent functions are ideally tailored to regulate mitochondria localization and activity in keratinocytes, very little is known about the contribution of keratins in the regulation of mitochondria in the epidermis. We have recently identified keratin isotypes as major regulators of desmosome adhesion through PKCalpha and as essential scaffolds for the epidermal barrier. Now, we found that mitochondrial distribution in keratinocytes is keratin-dependent. In keratin-deficient keratinocytes, increased levels of mitochondrial phosphatidyl-ethanolamine and cardiolipin and of electron transport chain complex 1 and 4 proteins were found, resulting in O2 consumption elevated by 30 %. Finally, keratin-deficient mice hair follicle morphogenetic defects, also reported in mice with an epidermal-specific deletion of the electron transport chain. Here, we want to understand how keratin isotypes regulate mitochondrial positioning, dynamics and physiology in keratinocytes and in vivo. To resolve this, we will 1) identify proteins which mediate keratin-mitochondria interactions and identify their interaction domains. In addition to the above candidate proteins, BioID will be used to identify novel interacting proteins. Interactions will be characterized by confocal imaging and protein interaction methods and verified by gain and-loss of function studies in our keratin-modulated keratinocytes. 2) We will analyse the role of normal and mutant keratins for mitochondria-ER interaction and for mitochondrial dynamics to understand how keratins control mitochondrial localization and physiology during differentiation and in keratinopathies. 3) We will investigate whether the above and/or additional proteins and mechanisms contribute to the defects in hair follicle morphogenesis observed in keratin-deficient mice. Our proposal will provide novel insights into coupling of mitochondria to epidermal differentiation by keratins. Moreover, our data will advance the mechanistic understanding of mitochondrial and keratin disorders and may offer novel approaches to restore mitochondrial integrity.