Keratin-dependent regulation of mitochondria in keratinocytes and mouse epidermis

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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.