Keratin-dependent regulation of mitochondria in keratinocytes and mouse epidermis

线粒体的完整性和功能取决于正确的定位、与内质网的相互作用和对其动力学的调节。线粒体定位和动力学的机制是由支架和调节蛋白协调的，它们介导与细胞骨架的相互作用。除了微管，中间丝蛋白desmin， vimentin和简单上皮角蛋白参与线粒体定位和形状，可能通过与线粒体相关蛋白plectin 1b， trichoplein和Pirh2的相互作用。我们对哺乳动物表皮的线粒体分布和调控知之甚少。最近的研究表明，线粒体ROS信号对表皮分化和毛囊形态发生至关重要，而不是ATP。虽然角蛋白细胞骨架蛋白，由于其同型多样性和环境依赖的功能，理想地调节线粒体定位和角化细胞中的活性，但对角蛋白在表皮线粒体调节中的作用知之甚少。我们最近发现角蛋白同型是通过PKCalpha调节桥粒粘附的主要调节因子，也是表皮屏障的必要支架。现在，我们发现角化细胞中的线粒体分布是依赖于角蛋白的。在角蛋白缺失的角质形成细胞中，发现线粒体磷脂酰乙醇胺和心磷脂以及电子传递链复合物1和4蛋白水平升高，导致氧气消耗增加30%。最后，角蛋白缺乏的小鼠毛囊形态发生缺陷，也报道了表皮特异性电子传递链缺失的小鼠。在这里，我们想了解角蛋白同型如何调节线粒体定位、动态和生理在角化细胞和体内。为了解决这个问题，我们将1)鉴定介导角蛋白-线粒体相互作用的蛋白质并鉴定其相互作用域。除了上述候选蛋白外，BioID还将用于鉴定新的相互作用蛋白。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化细胞的功能增益和损失研究来验证。2)我们将分析正常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朊病过程中如何控制线粒体定位和生理。3)我们将研究上述和/或其他蛋白质和机制是否有助于在角蛋白缺陷小鼠中观察到的毛囊形态发生缺陷。我们的建议将为线粒体与表皮分化的角蛋白耦合提供新的见解。此外，我们的数据将促进对线粒体和角蛋白疾病的机制理解，并可能提供恢复线粒体完整性的新方法。

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.