Regulation of keratin network dynamics by the mechanical properties of the environment in migrating cells.

Keratin intermediate filaments provide mechanical resilience for epithelia. They are nevertheless highly dynamic and turn over continuously, even in sessile keratinocytes. The aim of this study was to characterize and understand how the dynamic behavior of the keratin cytoskeleton is integrated in migrating cells. By imaging human primary keratinocytes producing fluorescent reporters and by using standardized image analysis we detect inward-directed keratin flow with highest rates in the cell periphery. The keratin flow correlates with speed and trajectory of migration. Changes in fibronectin-coating density and substrate stiffness induces concordant changes in migration speed and keratin flow. When keratinocytes are pseudo-confined on stripes, migration speed and keratin flow are reduced affecting the latter disproportionately. The regulation of keratin flow is linked to the regulation of actin flow. Local speed and direction of keratin and actin flow are very similar in migrating keratinocytes with keratin flow lagging behind actin flow. Conversely, reduced actin flow in areas of high keratin density indicates an inhibitory function of keratins on actin dynamics. Together, we propose that keratins enhance persistence of migration by directing actin dynamics and that the interplay of keratin and actin dynamics is modulated by matrix adhesions.

Hemidesmosomes and Focal Adhesions Treadmill as Separate but Linked Entities during Keratinocyte Migration.

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. New hemidesmosomal chevrons form subsequent to focal adhesion assembly at the cell's leading front, whereas chevrons and associated focal adhesions disassemble at the cell rear in reverse order. The bulk of the hemidesmosome-focal adhesion composite, however, remains attached to the substratum during cell translocation. Similar hemidesmosome-focal adhesion patterns emerge on X-shaped fibronectin-coated micropatterns, during cell spreading and in leader cells during collective cell migration. We further find that hemidesmosomes and focal adhesions affect each other's distribution. We propose that both junctions are separate but linked entities, which treadmill coordinately to support efficient directed cell migration and cooperate to coordinate the dynamic interplay between the keratin and actin cytoskeleton.

Facile and cost-effective production of microscale PDMS architectures using a combined micromilling-replica moulding (µMi-REM) technique.

We describe a cost-effective and simple method to fabricate PDMS-based microfluidic devices by combining micromilling with replica moulding technology. It relies on the following steps: (i) microchannels are milled in a block of acrylic; (ii) low-cost epoxy adhesive resin is poured over the milled acrylic block and allowed to cure; (iii) the solidified resin layer is peeled off the acrylic block and used as a mould for transferring the microchannel architecture onto a PDMS layer; finally (iv) the PDMS layer is plasma bonded to a glass surface. With this method, microscale architectures can be fabricated without the need for advanced technological equipment or laborious and time-consuming intermediate procedures. In this manuscript, we describe and validate the microfabrication procedure, and we illustrate its applicability to emulsion and microbubble production.