One step ahead: role of filopodia in adhesion formation during cell migration of keratinocytes.

Cell adhesion is an essential prerequisite for cell function and movement. It depends strongly on focal adhesion complexes connecting the extracellular matrix to the actin cytoskeleton. Especially in moving cells focal adhesions are highly dynamic and believed to be formed closely behind the leading edge. Filopodia were thought to act mainly as guiding cues using their tip complexes for elongation. Here we show for keratinocytes a strong dependence of lamellipodial adhesion sites on filopodia. Upon stable contact of the VASP-containing tip spot to the substrate, a filopodial focal complex (filopodial FX) is formed right behind along the filopodia axis. These filopodial FXs are fully assembled, yet small adhesions containing all adhesion markers tested. Filopodial FXs when reached by the lamellipodium are just increased in size resulting in classical focal adhesions. At the same time most filopodia regain their elongation ability. Blocking filopodia inhibits development of new focal adhesions in the lamellipodium, while focal adhesion maturation in terms of vinculin exchange dynamics remains active. Our data therefore argue for a strong spatial and temporal dependence of focal adhesions on filopodial focal complexes in keratinocytes with filopodia not permanently initiated via new clustering of actin filaments to induce elongation.

The secretory beta-amyloid precursor protein is a motogen for human epidermal keratinocytes.

Cell migration is known to be triggered by constituents of the extracellular matrix such as fibronectin and by soluble mediators commonly summarized as motogens. Many growth factors such as the epidermal growth factor (EGF) have been shown to act as motogens. Recently, the secretory N-terminal portion of the beta-amyloid precursor protein (sAPP) has been identified as a keratinocyte growth factor. Hence, in this study we analysed whether sAPP stimulates also keratinocyte migration employing the stroboscopic cell motility assay. The migration velocity as well as the frequency of lamellipodia protrusion and ruffle formation were increased about two-fold thus corresponding to the effect of EGF. Using a newly developed beta1-integrin migration track assay we observed that sAPP increased the proportion of migrating keratinocytes and their directional persistence. sAPP appeared to operate synergistically with fibronectin with respect to its motogenic effect. Using a modified Boyden chamber assay we showed that sAPP besides its chemokinetic effect functions as a chemoattractant. Like EGF, sAPP exerted its motogenic effect through the activation of Rac kinase but the receptor for sAPP appears to be distinct. The results suggest that sAPP operates as a motogen in the human epidermis, where it may participate in the regulation of reepithelialization during wound healing.

Cell migration: mechanisms of rear detachment and the formation of migration tracks.

Cell migration is central to many biological and pathological processes, including embryogenesis, tissue repair and regeneration as well as cancer and the inflammatory response. In general, cell migration can be usefully conceptualized as a cyclic process. The initial response of a cell to a migration-promoting agent is to polarize and extend protrusions in the direction of migration. These protrusions can be large, broad lamellipodia or spike-like filopodia, are usually driven by actin polymerization, and are stabilized by adhering to the extracellular matrix (ECM) via transmembrane receptors of the integrin family linked to the actin cytoskeleton. These adhesions serve as traction sites for migration as the cell moves forward over them, and they must be disassembled at the cell rear, allowing it to detach. The mechanisms of rear detachment and the regulatory processes involved are not well understood. The disassembly of adhesions that is required for detachment depends on a coordinated interaction of actin and actin-binding proteins, signaling molecules and effector enzymes including proteases, kinases and phosphatases. Originally, the biochemically regulated processes leading to rear detachment of migrating cells were thought not to be necessarily accompanied by any loss of cell material. However, it has been shown that during rear detachment long tubular extensions, the retracting fibers, are formed and that "membrane ripping" occurs at the cell rear. By this process, a major fraction of integrin-containing cellular material is left behind forming characteristic migration tracks that exactly mark the way a cell has taken.

Laminin-5-deficient human keratinocytes: defective adhesion results in a saltatory and inefficient mode of migration.

Laminin-5 is a major adhesion protein of the skin basement membrane and crucially involved in integrin-mediated cell substrate attachment of keratinocytes, which is important for hemidesmosomal anchorage as well as for keratinocyte migration during epidermal wound healing. To investigate its role in keratinocyte migration, we analyzed laminin-5-deficient cells of patients with a lethal variant of junctional epidermolysis bullosa. Normal migrating keratinocytes adopted monopolar morphology with a distinct front lamella and employed a continuous mode of translocation. In contrast, laminin-5-deficient cells assumed a stretched bipolar shape with two lamella regions and migrated in a discontinuous, saltatory manner characterized by significantly decreased directional persistence and reduced migration velocity. The distinct morphology as well as the migratory phenotype apparently resulted from a defect in the formation of cell substrate adhesions that were completely missing in the cell body and less stable in the lamella regions. Accordingly in normal keratinocytes, a bipolar shape and a saltatory migration mode were inducible by blocking laminin-5-mediated substrate adhesion. Our findings clearly point to an essential role of laminin-5 in forming dynamic cell substrate adhesion during migration of epidermal keratinocytes and provide an explanation for the cellular mechanisms that underlie the lethal form of junctional epidermolysis bullosa.

Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization.

During epithelial cell migration, membrane ruffles can be visualized by phase contrast microscopy as dark waves arising at the leading edge of lamellipodia that move centripetally toward the main cell body. Despite the common use of the term membrane ruffles, their structure, molecular composition, and the mechanisms leading to their formation remained largely unknown. We show here that membrane ruffles differ from the underlying cell lamella by more densely packed bundles of actin filaments that are enriched in the actin cross-linkers filamin and ezrin, pointing to a specific bundling process based on these cross-linkers. The accumulation of phosphorylated, that is, inactivated, cofilin in membrane ruffles suggests that they are compartments of inhibited actin filament turnover. High Rac1 and low RhoA activities were found under conditions of suboptimal integrin-ligand interaction correlating with low lamellipodia persistence, inefficient migration, and high ruffling rates. Based on these findings, we define membrane ruffles as distinct compartments of specific composition that form as a consequence of inefficient lamellipodia adhesion.

CD83 localization in a recycling compartment of immature human monocyte-derived dendritic cells.

Dendritic cells (DC) change their phenotype and functional properties during maturation. CD83 cell surface expression is induced on mature DC (mDC). In this study, we investigated intracellular CD83 localization and transport in human monocyte-derived DC. The enhanced level of CD83 cell surface expression in mDC resulted predominantly from increased protein synthesis, and in addition from regulated intracellular transport of CD83 protein. An internal pool of CD83 protein is present in immature DC (iDC). Although CD83 protein in iDC and in mDC was localized in the Golgi compartment and in recycling endosomes, only in mature cells did CD83 co-localize with MHC class II molecules in endocytic vesicles. CD83 cell surface expression on iDC was induced by inhibition of endocytosis. This result could be explained by CD83 cycling between endosomes and the cell surface in iDC. The mDC also rapidly internalized membrane-bound CD83 protein. Furthermore, a thiol protease inhibitor and specific cathepsin inhibitors impaired CD83 up-regulation in DC, indicating a role of endosomal proteases in the maturation-induced exposure of CD83 on the plasma membrane.

Structural and compositional analysis of the keratinocyte migration track.

Slowly migrating cells such as fibroblasts leave behind a "migration track," which has been assumed not to occur in fast-moving cells such as keratinocytes. Here we show that keratinocytes left behind "migration tracks" of cellular remnants consisting of membranous patches or macroaggregates that were anchored to a meshwork of extracellular matrix proteins consisting of collagen type IV, fibronectin, laminin, and laminin 5. According to their origin and localisation, two types of macroaggregates could be distinguished : (1) Spherical and elongated tubular structures (diameter about 50-110 nm) both of which were arranged like "pearls on a string" and that apparently derived from fragmentation of retracting fibres. (2) Spherical structures (diameter about 50 nm) left behind in the gaps between the retracting fibres and presumably derived from former focal adhesion sites. Both types of macroaggregates did not contain cytoplasmic proteins but carried on their surface adhesion proteins, particularly high amounts of integrins : type 1 macroaggregates contained alpha3beta1-integrins, whereas type 2 macroaggregates contained other types of integrins such as alpha6beta4-integrins. Modulation of keratinocyte adhesion by using poly-L-lysine coated cover slips resulted in an increased application of inhibitory beta1-antibodies and slightly reduced migration velocity and track formation. Within 24 h of migration, we observed a migration velocity-dependent loss of cellular beta1-integrin by macroaggregate formation of about 11% for fast and about 4% for slowly migrating keratinocytes. The physiological role of the migration track is unclear. However, with its multiple adhesion sites it may serve as a provisional basement membrane during reepithelialization of epidermal wounds.