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) demonstrate that in migrating primary human keratinocytes, hemidesmosomes cluster as ordered arrays consisting of multiple chevrons, flanked by actin-associated focal adhesions. These and related findings have implications for wound healing, cancer invasion, blistering skin diseases, and skin aging.
Epidermal keratinocytes tightly interact with the extracellular matrix along their basal membrane through integrin transmembrane proteins organized into α3β1-enriched focal adhesions (FA) and α6β4-positive hemidesmosomes (HD). Inside the cell, these integrins attach to and regulate the actin and keratin cytoskeletons, respectively, via a multitude of scaffold and regulatory proteins (
). Firm attachment and dynamic regulation via FA and HD are essential to maintain major skin functions, such as self-renewal, barrier function, and resilience to physical and chemical stresses. Conversely, dysregulation of both basement membrane junctions by mutations in corresponding genes, autoantibodies, or environmental insults, such as UV damage or viral infections, can cause a wide range of pathologies, including epidermolysis bullosa, pemphigus, wound healing defects, cancer and skin aging (
Although much has already been learned about the composition and functions of FA, our knowledge of HD and their constituent proteins, as well as their respective contributions to cell migration and adhesion, is still incomplete. FA are bidirectional hubs that coordinate inside-out and outside-in chemical and mechanical signaling and promote migration and dynamic adhesion. These complex roles are reflected by an extensive number of constituents (
). In contrast, the understudied proteome of HD currently comprises ≤10 known proteins and HD have been viewed as mainstays of stable adhesion rather than as signaling hubs, although the latter view has been challenged by several reports (
In the past, several studies have shown an interdependence of FA and HD, using mostly immortalized cells. However, the principles governing the coordination of FA and HD during keratinocyte differentiation and migration remain incompletely understood. Using high resolution imaging, Pora and colleagues (
) describe highly ordered, interdigitated arrays of chevron patterns of HD and FA in primary human keratinocytes that seem to depend on each other to sustain directed collective migration. In primary keratinocytes migrating on fibronectin, these HD extend from the rear to the lamellipodial cell front. Staining for the major known constituent proteins α6β4, BP230, and plectin and for the associated keratins supports their characterization as HD. Paxillin staining revealed that actin-associated FA interdigitated with HD chevrons but never showed exact overlap, irrespective of the shape of substrata used.
To study the interdependence of FA and HD, the authors analyzed migrating keratinocytes transfected with autofluorescent paxillin and β4 integrin. As expected, nascent FA appeared first, oriented pairwise in oblique angles to the direction of migration, and became larger at the lamellum–lamellipodium interface. Subsequently, hemidesmosomal β4 integrin accumulated in the space between FA and extended the chevron-like structures toward cell fronts. Once established, patterned HD and FA remained stationary with respect to the substrate without positional changes of the corresponding components. In contrast,
found movements of FA and HD with respect to the substrate in immortalized human keratinocytes and rat urothelial cells. This apparent discrepancy may result from distinct post-translational modifications prevailing in these two cell types.
The continued and coordinated growth of HD–FA chevrons at cell fronts subsequently resulted in the translocation of cell bodies toward cell fronts. At cell rear, old FA were removed just prior to retraction fiber formation, whereas β4-positive structures remained attached until the retraction fibers were severed, causing accumulation of substrate-attached extracellular β4-positive patches. The chevron-like, interdigitated arrangement of HD and FA also prevailed in the setting of cell spreading. Here, precursors of keratin filaments emerged close to the FA in extreme cell peripheries, as described before (
), whereas filaments were found at β4 dots. By limiting FA formation with blocking β1 antibodies, the authors examined the consequences for HD. As one might have expected, FA were smaller and remained at cell peripheries, surrounded by HD, which were not only reduced in size, but had also lost their chevron-like appearance. Conversely, applying α6-blocking antibodies significantly reduced the sizes of both HD and FA, supporting the interdependence of both junctions.
In view of the known interactions of α3β1 and α6β4 with laminin-332, the authors next investigated the contribution of laminin-332, showing that it accelerated keratinocyte migration when offered as a substrate instead of fibronectin. How FA and HD contribute to this effect remains to be investigated. Finally, the authors asked whether the chevron-like array of epidermal FA and HD bears significance for wound healing and cancer cell metastasis by examining collective cell migration in a dedicated in vitro assay. Most notably, leader cells formed typical intercalating FA-HD chevron patterns, whereas follower cells did not, supporting the view that mechanical signals emanating from FA facilitate HD formation (
). Their apparent absence in confluent monolayers suggests that FA-HD chevrons are transient structures critical in distinct stages of adhesion and migration.
Collectively, the above data substantiate and extend previous findings of highly ordered and interdependent formation of FA and HD in several types of epithelial cells that is required to sustain directed migration (
). To explain their findings, the authors discuss potential mechanisms that could underlie the spatiotemporal assembly of the two cell-matrix junctions at several levels: scaffold proteins such as plectin or acto-myosin fibers could provide a mechanical linkage; steric facilitation could bring the FA-adjacent plasma membrane in close apposition to the ECM to promote laminin-332 deposition and α6β4 clustering; tension elicited by acto-myosin fibers or by mechanosensing proteins in FA could initiate HD protein deposition in their vicinity. Conversely, Rac1 signaling downstream of α6β4 is known to affect FA integrin composition in a feedback mechanism (
), much less is known about HD regulation. The significant loss of HD integrins together with laminin-332 at the cell rears requires newly synthesized α6β4 integrin for HD formation and cell migration (
) (see Figure 1). Since mRNAs encoding HD proteins are large (e.g., 5860 bp for α6 integrin), de novo transcription and translation is unlikely to provide enough protein to allow for the observed migration velocities. Post-transcriptional regulation of mRNA stability and/or translational efficiency is more likely to support HD formation. Interestingly, α6β4 heterodimer formation and α6 localization were found to depend on the α6 3’UTR (
). Moreover, a role for 3’UTR in membrane protein localization has recently emerged as a more common principle, since CD47, CD44, ITGA1 and TNFRSF13C mRNAs were all bound by the RNA-binding protein HuR/ELAVL1 to increase cell surface expression without altering mRNA localization or protein levels (
). Instead, an adapter protein recruited by ELAVL mediated the correct localization. Interestingly, β4 interacts with several RNA-binding proteins comprising well-known regulators of translation and AU-rich element binding proteins. This highlights the significance of post-transcriptional regulation in controlling cell migration and points to the topic of integrin/integrin pair sorting in the ER and in vesicular compartments to reach their respective compartments. An attractive hypothesis is that adapter proteins interacting with integrin mRNAs could support specific dimer formation.
Several findings point to a major role for keratins in connecting FA and HD and in stabilizing the latter adhesion complexes. In their previous work, the Leube group has provided strong evidence for the emergence of assembly-competent keratin filament precursors next to established FA, using high resolution live cell imaging (
) (Figure 1). Subsequently, these precursors grow in length while moving away from FA. Even though the mode of transport of growing keratin filament precursors is not yet clear, it has been demonstrated that keratin filaments are essential for the maintenance of stable HD by interacting with plectin, in addition to BP230 (
). The concept that reduction or loss of keratins not only destabilizes HD but enhances collective cell migration supports a role of the HD-keratin complex in cell-matrix adhesion over migration. Additionally, keratins can control FA and HD indirectly by virtue of their interactions with Src and myoIIA. Collectively, these emerging data point to a prominent involvement of keratins in the interdependence of HD and FA, which is probably more complex than currently appreciated.
The increased complexity of disease mechanisms for which FA-HD interdependence serves as a good example is underscored by the recently discovered involvement of HD in skin aging, a process that depends on stem cell maintenance (
). The dynamic interaction of keratinocyte stem cells with their ECM niche requires many factors, including col XVII/BP180. Recently, Liu and colleagues showed that col XVII/BP180 levels were reduced by genomic/oxidative stress, leading to fewer and less adhesive HD. This novel link between HD and genomic stress via col XVII profoundly affects cell turnover in the epidermis. Clones that express high levels of COL17A1/BP180 and divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1/BP180, which divide asymmetrically. This leads to the selection of stem cells with higher potential or quality. However, ultimately, their loss of COL17A1/BP180 limits their competition, contributing to skin aging. Thus, fine-tuning cell-matrix adhesion by a multitude of mechanisms is crucial for the maintenance of adhesion, and also lends itself to potential therapeutic intervention.
Hemidesmosomes anchor the epidermal keratin filament cytoskeleton to the extracellular matrix. They are crucial for the mechanical integrity of skin. Their role in keratinocyte migration, however, remains unclear. Examining migrating primary human keratinocytes, we find that hemidesmosomes cluster as ordered arrays consisting of multiple chevrons that are flanked by actin-associated focal adhesions. These hemidesmosomal arrays with intercalated focal adhesions extend from the cell rear to the cell front.