Impact of keratin network regulation on migrating cells

Cell migration is a highly complex process whereby different physical and chemical signals act on many different cell components, most notably the force-generating cytoskeleton. Intermediate filaments are one of the three major components of the cytoskeleton. Keratin intermediate filaments are the main type of cytoplasmic intermediate filaments of epithelial cells. They are anchored to hemidesmosomes, which are involved in the attachment of epithelial cells to the extracellular matrix of the underlying basement membrane. Although keratin intermediate filaments are involved in the mechanical resilience of tissues, they are highly dynamic structures. They are subject to continuous turnover in sessile keratinocytes. This turnover is part of a spatially-defined cycle of assembly and disassembly. Similarly, hemidesmosomes contribute to the mechanical integrity of the epithelium, but their role in epithelial cell migration remains unclear. I aimed at understanding how the dynamic behavior of the epithelial keratin intermediate filament cytoskeleton and its associated hemidesmosomes are integrated in migrating cells. Most particularly, I investigated how the distribution and the kinetics of the keratin turnover cycle are affected by cell migration; and how this process is dependent on the mechanophysical environment of the cell. I determined how hemidesmosomes are organized and maintained during migration; and how this process depends on the adjacent actin-associated focal adhesions. In migrating human primary keratinocytes, I show that keratin is highly dynamic presenting a well-defined spatial distribution with highest flow found at the cell periphery. Keratin dynamics are co-regulated with the speed of cell migration and the cell’s trajectory. Upon changes in the mechanophysical environment, migration features and keratin flow are altered. The changes in keratin flow are most likely downstream of focal adhesion-dependent mechanosensation and actin flow. As a result, keratin dynamics correlate with migration speed. Keratin flow and actin flow show conspicuous similarities in their spatial distribution. The observed dynamic interplay suggests the existence of multiple feedbacks between the actin and keratin cytoskeleton. My observations further suggest that the associated cell-matrix adhesions play a key role in this feedback. I then show that in migrating cells, hemidesmosomes cluster into highly ordered chevronlike patterns with intercalated focal adhesions in migrating primary human keratinocytes. This arrangement is maintained during migration by continuous assembly of adhesions at the cell front and disassembly of adhesions at the cell back. I further observed that the specialized hemidesmosome-focal adhesion distribution patterns emerge during substrate adhesion and cell spreading within three hours after seeding. During cell adhesion and migration, hemidesmosomes and focal adhesions affect each other’s distribution. Bidirectional cross-talk between focal adhesions and hemidesmosomes is key for the coordination of the actin and keratin cytoskeleton during cell migration. Mechanical coupling at the cell-matrix adhesion level and at the cytoskeletal network level together induce an increase in migratory persistence. Persistence in cell migration is crucial for efficient migration in a complex 3D environment

Facile and cost-effective production of microscale PDMS architectures using a combined micromilling-replica moulding (uMi-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

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

Genomic and Molecular Landscape of DNA Damage Repair Deficiency across The Cancer Genome Atlas

Summary: DNA damage repair (DDR) pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying loss of heterozygosity were observed in over 1/3 of DDR genes, including TP53 and BRCA1/2. Other prevalent alterations included epigenetic silencing of the direct repair genes EXO5, MGMT, and ALKBH3 in ∼20% of samples. Homologous recombination deficiency (HRD) was present at varying frequency in many cancer types, most notably ovarian cancer. However, in contrast to ovarian cancer, HRD was associated with worse outcomes in several other cancers. Protein structure-based analyses allowed us to predict functional consequences of rare, recurrent DDR mutations. A new machine-learning-based classifier developed from gene expression data allowed us to identify alterations that phenocopy deleterious TP53 mutations. These frequent DDR gene alterations in many human cancers have functional consequences that may determine cancer progression and guide therapy. : Knijnenburg et al. present The Cancer Genome Atlas (TCGA) Pan-Cancer analysis of DNA damage repair (DDR) deficiency in cancer. They use integrative genomic and molecular analyses to identify frequent DDR alterations across 33 cancer types, correlate gene- and pathway-level alterations with genome-wide measures of genome instability and impaired function, and demonstrate the prognostic utility of DDR deficiency scores. Keywords: The Cancer Genome Atlas PanCanAtlas project, DNA damage repair, somatic mutations, somatic copy-number alterations, epigenetic silencing, DNA damage footprints, mutational signatures, integrative statistical analysis, protein structure analysi