The keratin-filament cycle of assembly and disassembly

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Keratin intermediate filaments are major constituents of the epithelial cytoskeleton. Their organizing function of cellular space is important for diverse epithelial functions requiring a high degree of keratin network plasticity. We have recently proposed that a continuous cycle of keratin filament assembly and disassembly affords rapid and precise adaptation to the different functional requirements and situations. The goal of the first project phase was to establish and apply tools for proving/disproving our concept of the keratin cycle and to examine its proposed aspects. Our experiments observing fluorescently-tagged keratin polypeptides fully supported the main features of the keratin cycle. Furthermore, they allowed us to quantitatively measure keratin network properties, speed of keratin movement and local turnover. We were further able to demonstrate that the new tools are capable detecting slight differences in keratin cycling taking place after seeding of cells and upon growth factor addition, migration induction, keratin phosphorylation, and downregulation of cytoskeletal cross linkers as well as in response to altered substrate stiffness. The second project phase intends to move from a descriptive stage of the keratin cycle to a molecular understanding. To achieve this, we will (i) further refine methods and tools for monitoring and manipulating the keratin cycle and its modulators, (ii) focus on identifying and characterizing the molecular motors involved in keratin movement and tension generation within the keratin network, and (iii) examine modulators of the keratin cycle that are identified in genetic screens. We will apply the newly developed image recording and analysis routines on high resolution microscopic image sets of improved fluorescent reporter cell lines to systematically study the effect of downregulating all of the relevant motor proteins and other modulators of the keratin cycle on keratin network organization, dynamics and function. Consequences on epithelial function, i.e. cell migration, cellular viscoelasticity and stiffness, proliferation and stress response will be correlated to these findings.Our long-term goal is to understand molecular mechanisms of keratin network plasticity in the context of epithelial pathophysiology.