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.

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.

Actin filament and microtubule growth characteristics are defined by their different plus and minus ends. In contrast, intermediate filaments lack this type of polarity. Yet, intermediate filament network growth occurs by selective addition of newly formed and polymerizing keratin particles at peripheral network domains thereby allowing polarized network reorganization. To examine this process at high resolution in living cells, mammary epithelium-derived, immortalized EpH4-cells were infected with retroviral cDNA constructs coding for human keratin 18-fluorescent protein hybrids. Several stable cell lines were established presenting characteristic fluorescent keratin filament (KF) networks. These cells contain particularly large and abundant lamellipodia in which nascent keratin particle dynamics are easily detected by time-lapse fluorescence microscopy. These keratin particles originate close to the plasma membrane, translocate continuously toward the cell center, and integrate end-on into the peripheral KF network. We show that this inward-directed transport relies on intact actin filaments. After treatment with the actin filament-disrupting drug cytochalasin newly polymerizing keratin assemblies still appear in the peripheral cytoplasm but remain stationary. On the other hand, nocodazole-mediated disruption of microtubules does not affect the centripetal KF precursor transport. From these and other observations a model is deduced which postulates that focal adhesion-dependent keratin polymerization occurs in forming lamellipodia and that transport of newly formed keratin particles is mediated by actin filaments until network integration. This mechanism allows extension of the KF network toward the leading edge in migrating cells and may be of relevance for tissue development and regeneration.

CYCLOIDEA-like genes belong to the TCP family of transcriptional regulators and have been shown to control different aspects of shoot development in various angiosperm lineages, including flower monosymmetry in asterids and axillary meristem growth in monocots. Genes related to the CYC gene from ANTIRRHINUM show independent duplications in both asterids and rosids. However, it remains unclear to what extent this affected the evolution of flower symmetry and shoot branching in these and other eudicot lineages. Here, we show that CYC-like genes have also undergone duplications in two related Ranunculales families, Fumariaceae and Papaveraceae s.str. These families exhibit morphological diversity in flower symmetry and inflorescence architecture that is potentially related to functions of CYC-like genes. We present sequences of 14 CYC-related genes covering 9 genera. Phylogenetic analyses indicate the presence of three clades of CYC-like genes. Shared motifs in the region between the TCP and R domains of CYC-like genes between Fumariaceae, Papaveraceae s.str., and AQUILEGIA (Ranunculaceae) indicate that the observed duplications originated from a single CYC gene present in all Ranunculales. RT-PCR expression data suggest that gene duplication and diversification in Fumariaceae and Papaveraceae s.str. was accompanied by divergence in expression patterns.

Peltate leaf architecture has evolved from conventional bifacial leaves many times in flowering plant evolution. Characteristics of peltate leaves, such as the differentiation of a cross zone and of a radially symmetric, margin-less petiole, have also been observed in mutants of genes responsible for adaxial-abaxial polarity establishment. This suggests that altered regulation of such genes provided a mechanism for the evolution of peltate leaf structure. Here, we show that evolution of leaf peltation in Tropaeolum majus, a species distantly related to Arabidopsis thaliana, was associated with altered expression of Tropaeolum majus FILAMENTOUS FLOWER (TmFIL), a gene conferring abaxial identity. In situ hybridization indicates that adaxial and abaxial domains are established in early leaf primordia as in species with bifacial leaves. Upon initiation of the cross zone by fusion of the blade margins, localized expansion of TmFIL to the upper leaf side could be seen, indicating a local loss of adaxial leaf identity. The observed changes in expression are consistent with a role of TmFIL in radialization of the petiole and circularization of the leaf blade margin by the cross zone. In addition, expression was observed in segment primordia and during expansion of the bifacial blade, suggesting additional roles for TmFIL in leaf development.