Keratin-dependent regulation of mitochondria in keratinocytes and mouse epidermis

线粒体的完整性和功能性取决于正确的定位、与ER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节蛋白介导的细胞骨架的相互作用。除了微管，中间丝蛋白结蛋白，波形蛋白和简单的上皮角蛋白参与线粒体的定位和形状，可能通过与线粒体相关的蛋白质plectin 1b，plecplein和Pirh 2的相互作用。目前对哺乳动物表皮线粒体的分布和调控知之甚少。最近的研究表明，线粒体ROS信号而不是ATP是至关重要的表皮分化和毛囊形态发生。虽然角蛋白细胞骨架蛋白，由于其同种型多样性和上下文依赖的功能是理想的定制，以调节角质形成细胞中的线粒体定位和活性，很少有人知道角蛋白在调节表皮中的线粒体的贡献。我们最近已经确定角蛋白同种型作为桥粒粘附通过PKCalpha的主要调节剂和作为表皮屏障的必要支架。现在，我们发现角质形成细胞中的线粒体分布是依赖于角蛋白的。在角蛋白缺乏的角质形成细胞，线粒体磷脂酰乙醇胺和心磷脂和电子传递链复合物1和4蛋白质的水平增加，导致O2消耗升高30%。最后，角蛋白缺乏的小鼠毛囊形态发生缺陷，也报告在小鼠表皮特异性缺失的电子传递链。在这里，我们想了解角蛋白同种型如何调节角质形成细胞和体内线粒体的定位，动力学和生理学。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它们的相互作用结构域。除了上述候选蛋白，BioID将用于识别新的相互作用蛋白。通过共聚焦成像和蛋白质相互作用方法表征相互作用，并通过我们的角蛋白调节的角质形成细胞的功能研究的获得和丧失来验证。2)我们将分析正常和突变的角蛋白的作用，ESTA-ER的相互作用和线粒体动力学，以了解角蛋白如何控制分化过程中的线粒体定位和生理学，在角蛋白病。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。我们的建议将提供新的见解耦合线粒体表皮分化角蛋白。此外，我们的数据将推进线粒体和角蛋白疾病的机制理解，并可能提供新的方法来恢复线粒体的完整性。

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.