Reinhard Windoffer

Structure and Function of Desmosomes

Desmosomes are prominent adhesion sites that are tightly associated with the cytoplasmic intermediate filament cytoskeleton providing mechanical stability in epithelia and also in several nonepithelial tissues such as cardiac muscle and meninges. They are unique in terms of ultrastructural appearance and molecular composition with cell type-specific variations. The dynamic assembly properties of desmosomes are important prerequisites for the acquisition and maintenance of tissue homeostasis. Disturbance of this equilibrium therefore not only compromises mechanical resilience but also affects many other tissue functions as becomes evident in various experimental scenarios and multiple diseases.

Loss of desmoglein 2 suggests essential functions for early embryonic development and proliferation of embryonal stem cells

Desmoglein 2 (Dsg2) is a Ca(2+)-dependent adhesion molecule of desmosomes and is synthesized in all desmosome-bearing tissues from their earliest appearance onward. To examine the function of Dsg2, its gene was inactivated by homologous recombination in embryonal stem (ES) cells for the generation of knockout mice. DSG2 -/- mice and a considerable number of DSG2 +/- mice died at or shortly after implantation. On the other hand, DSG2 -/- blastocysts developed an apparently normal trophectoderm layer, the first tissue known to produce desmosomes, and hatched properly. Immunofluorescence analyses of these blastocysts showed, however, that the distribution of the desmosomal plaque protein desmoplakin was disturbed, whereas the adherens junction proteins E-cadherin and beta-catenin appeared to be unaffected. Unexpectedly, we found that Dsg2 seems to be essential for the inner cell mass and the ES cell population derived there from. We present evidence that Dsg2, which is located in desmoplakin-negative wild-type ES cells in non-desmosomal junctions, is needed for normal ES cell proliferation. Our observations thus reveal that important Dsg2 functions are desmosome-independent during early development and are needed for ES cell and early embryo survival.

Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia

Epithelia are exposed to multiple forms of stress. Keratin intermediate filaments are abundant in epithelia and form cytoskeletal networks that contribute to cell type–specific functions, such as adhesion, migration, and metabolism. A perpetual keratin filament turnover cycle supports these functions. This multistep process keeps the cytoskeleton in motion, facilitating rapid and protein biosynthesis–independent network remodeling while maintaining an intact network. The current challenge is to unravel the molecular mechanisms underlying the regulation of the keratin cycle in relation to actin and microtubule networks and in the context of epithelial tissue function.

Keratins regulate protein biosynthesis through localization of GLUT1 and -3 upstream of AMP kinase and Raptor

Removal of the entire keratin family of intermediate filament proteins from embryonic epithelia has surprising implications for mTOR signaling.

Keratins as the main component for the mechanical integrity of keratinocytes

Significance For decades, researchers have been trying to unravel one of the key questions in cell biology regarding keratin intermediate filament function in protecting epithelial cells against mechanical stress. For many different reasons, however, this fundamental hypothesis was still unproven. Here we answer this pivotal question by the use of keratin KO cells lacking complete keratin gene clusters to result in total loss of keratin filaments. This lack significantly softens cells, reduces cell viscosity, and elevates plastic cell deformation on force application. Reexpression of single keratin genes facilitates biomechanical complementation of complete cluster loss. Our manuscript therefore makes a very strong case for the crucial contribution of keratins to cell mechanics, with far-reaching implications for epithelial pathophysiology. Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.

Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases.

The cytokeratin filament network is intrinsically dynamic, continuously exchanging subunits over its entire surface, while conferring structural stability on epithelial cells. However, it is not known how cytokeratin filaments are remodeled in situations where the network is temporarily and spatially restricted. Using the tyrosine phosphatase inhibitor orthovanadate we observed rapid and reversible restructuring in living cells, which may provide the basis for such dynamics. By examining cells stably expressing fluorescent cytokeratin chimeras, we found that cytokeratin filaments were broken down and then formed into granular aggregates within a few minutes of orthovanadate addition. After drug removal, gradual reincorporation of granules into the filament network was observed for aggregates that were either part of residual filaments or stayed in close apposition to remaining filaments. Even when cytokeratin filaments were no longer detectable, granules with low mobility were still able to reestablish a cytokeratin filament network. This process took less than 30 minutes and occurred at multiple foci throughout the cytoplasm without apparent correlation to alterations in the actin- and tubulin-based systems. Interestingly, the short-lived and rather small orthovanadate-induced cytokeratin granules contained the cytoskeletal crosslinker plectin but lacked the cytokeratin-solubilising 14-3-3 proteins. By contrast, the long-lived and larger cytokeratin aggregates generated after treatment with the serine/threonine phosphatase inhibitor okadaic acid were negative for plectin but positive for 14-3-3 proteins. Taken together, our observations in living orthovanadate-treated interphase cells revealed modes of cytokeratin remodeling that qualify as basic mechanisms capable of rapidly adapting the cytokeratin filament cytoskeleton to specific requirements.

Keratins control intercellular adhesion involving PKC-α–mediated desmoplakin phosphorylation

Keratins limit PKC-α phosphorylation activity and desmosome turnover to ensure the stability of epithelial intracellular adhesion.

Focal adhesions are hotspots for keratin filament precursor formation

Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement.

p38 MAPK-dependent shaping of the keratin cytoskeleton in cultured cells

Plasticity of the resilient keratin intermediate filament cytoskeleton is an important prerequisite for epithelial tissue homeostasis. Here, the contribution of stress-activated p38 MAPK to keratin network organization was examined in cultured cells. It was observed that phosphorylated p38 colocalized with keratin granules that were rapidly formed in response to orthovanadate. The same p38p recruitment was noted during mitosis, in various stress situations and in cells producing mutant keratins. In all these situations keratin 8 became phosphorylated on S73, a well-known p38 target site. To demonstrate that p38-dependent keratin phosphorylation determines keratin organization, p38 activity was pharmacologically and genetically modulated: up-regulation induced keratin granule formation, whereas down-regulation prevented keratin filament network disassembly. Furthermore, transient p38 inhibition also inhibited keratin filament precursor formation and mutant keratin granule dissolution. Collectively, the rapid and reversible effects of p38 activity on keratin phosphorylation and organization in diverse physiological, stress, and pathological situations identify p38-dependent signalling as a major intermediate filament–regulating pathway.

The keratin-filament cycle of assembly and disassembly

Continuous and regulated remodelling of the cytoskeleton is crucial for many basic cell functions. In contrast to actin filaments and microtubules, it is not understood how this is accomplished for the third major cytoskeletal filament system, which consists of intermediate-filament polypeptides. Using time-lapse fluorescence microscopy of living interphase cells, in combination with photobleaching, photoactivation and quantitative fluorescence measurements, we observed that epithelial keratin intermediate filaments constantly release non-filamentous subunits, which are reused in the cell periphery for filament assembly. This cycle is independent of protein biosynthesis. The different stages of the cycle occur in defined cellular subdomains: assembly takes place in the cell periphery and newly formed filaments are constantly transported toward the perinuclear region while disassembly occurs, giving rise to diffusible subunits for another round of peripheral assembly. Remaining juxtanuclear filaments stabilize and encage the nucleus. Our data suggest that the keratin-filament cycle of assembly and disassembly is a major mechanism of intermediate-filament network plasticity, allowing rapid adaptation to specific requirements, notably in migrating cells.