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

Abstract

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 fibronectincoating 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.Cell migration is a highly complex process with relevance for physiological and pathological situations such as embryogenesis, wound healing and metastasis 1 . It is induced by chemical and biophysical cues. The cytoskeleton is at the core of generating effective locomotion by enabling successive steps of protrusion at the cell front and by facilitating the contractile events at the cell rear in parallel with the regulation of cell-matrix adhesions 2 . The cytoskeleton is composed of three major components, namely actin filaments, microtubules and intermediate filaments. The role of actin filaments and microtubules in cell migration has been extensively studied. In contrast, the contribution of intermediate filaments and especially of epithelial keratin intermediate filaments is still poorly understood 3 .Keratin filaments are the main class of cytoplasmic intermediate filaments in epithelial cells. They belong to a large multigene family encoded by more than 50 genes. Every keratin filament contains equal amounts of type I (acidic) and type II (basic) monomers. Obligatory heterodimers assemble in an antiparallel manner into non-polar tetramers, which further associate laterally and longitudinally to eventually generate 8-12 nm filaments 4-6 .The effect of keratin expression on migration depends on the keratin isoform, the cell type and the environment 3,7,8 . For example, the knockdown of K8/K18 reduces the invasion capacity of squamous cancer cells 9 but increases collective cell migration of cancer cells 10 . Depletion of the entire type II keratin gene cluster in mouse keratinocytes prevents keratin filament assembly and induces an increase in migration speed and invasiv...

Hemidesmosome-Related Keratin Filament Bundling and Nucleation

“…During migration of the cells, new focal adhesions and hemidesmosomes were formed at the leading edges and were disassembled at the trailing edges as previously described for migrating primary keratinocytes [ 29 ]. At the same time, new keratin filaments were formed preferentially at the leading edge that subsequently gained contact to the main cytoskeletal keratin network ( Figure 7 and corresponding Video S7 ; also see References [ 44 , 51 ]). Shortly before the appearance of keratin particles, integrin β4-YFP clusters could be detected at the same positions.…”

“…But an intricate interplay between focal adhesions and hemidesmosomes appears to take place during migration, which is of relevance during wound healing in human skin [ 29 , 31 , 32 , 37 ]. Even less is known about the coordination of the ECM-dependent organization of the different cytoskeletal systems although ECM-dependent coordinated regulation of cytoskeletal network dynamics in epithelial cells has been reported [ 43 , 44 ]. Most recently, a force-dissipating function has been assigned to hemidesmosomes and their associated keratin filaments by demonstrating that prevention of α6β4 adhesion increases focal adhesion-mediated and actin-dependent traction force generation in human keratinocytes [ 45 ].…”

Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy

“…KIFs are classified as type I and type II Intermediate Filament (IF) proteins, according to their sequence ( Szeverenyi et al, 2008 ; Bragulla and Homberger, 2009 ; Toivola et al, 2015 ). Keratins form a highly flexible and dynamic filamentous network in the cytoplasm ( Etienne-Manneville, 2018 ; Pora et al, 2020 ; Kölsch et al, 2010 ; Robert et al, 2016 ; Windoffer et al, 2004 ; Yoon et al, 2001 ). Their main known function is to protect the cell from external stresses by providing mechanical stability and ensuring the integrity of tissues through cell-cell and cell-matrix contacts.…”

Sp1-Induced FNBP1 Drives Rigorous 3D Cell Motility in EMT-Type Gastric Cancer Cells

“…Consistent with the gene ontology analysis, the gene set related to the anchored component of the plasma membrane was significantly enriched in MKN1 control cells compared to FNBP1 knockdown cells in gene set enrichment analyses (GSEA) (Figure 2E). Given that FNBP1 is expressed in EMT GC cell lines with high invasive capacity, GSEA also revealed changes in cell motility-related pathways (Figure 2E) [27,28].…”