Abstract
The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.
Highlights
The keratin cytoskeleton is a hallmark feature of epithelial cells [1,2]
It consists of a filamentous cytoplasmic network with unique biomechanical properties that is connected to desmosomes at cell-cell adhesion sites and to hemidesmosomes at epithelial-extracellular matrix (ECM) interfaces [3,4,5,6]
The current study focuses on keratin network organization in relation to hemidesmosomal adhesion examining the consequences of interfering with focal adhesion, actin filament polymerization and microtubule formation on keratin-hemidesmosome interaction
Summary
The keratin cytoskeleton is a hallmark feature of epithelial cells [1,2] It consists of a filamentous cytoplasmic network with unique biomechanical properties that is connected to desmosomes at cell-cell adhesion sites and to hemidesmosomes at epithelial-extracellular matrix (ECM) interfaces [3,4,5,6]. Its 3D organization and dynamic features rely on the other two major cytoskeletal filament systems, i.e., the actin-based microfilaments, which are anchored to adherens junctions at cell-cell borders and to focal adhesions at cell-ECM contacts, and the tubulin-based microtubules [7,8,9,10,11]. The current study focuses on keratin network organization in relation to hemidesmosomal adhesion examining the consequences of interfering with focal adhesion, actin filament polymerization and microtubule formation on keratin-hemidesmosome interaction
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