Actin‐dependent dynamics of keratin filament precursors

Abstract

Actin filament and microtubule growth characteristics are defined by their different plus and minus ends. In contrast, intermediate filaments lack this type of polarity. Yet, intermediate filament network growth occurs by selective addition of newly formed and polymerizing keratin particles at peripheral network domains thereby allowing polarized network reorganization. To examine this process at high resolution in living cells, mammary epithelium-derived, immortalized EpH4-cells were infected with retroviral cDNA constructs coding for human keratin 18-fluorescent protein hybrids. Several stable cell lines were established presenting characteristic fluorescent keratin filament (KF) networks. These cells contain particularly large and abundant lamellipodia in which nascent keratin particle dynamics are easily detected by time-lapse fluorescence microscopy. These keratin particles originate close to the plasma membrane, translocate continuously toward the cell center, and integrate end-on into the peripheral KF network. We show that this inward-directed transport relies on intact actin filaments. After treatment with the actin filament-disrupting drug cytochalasin newly polymerizing keratin assemblies still appear in the peripheral cytoplasm but remain stationary. On the other hand, nocodazole-mediated disruption of microtubules does not affect the centripetal KF precursor transport. From these and other observations a model is deduced which postulates that focal adhesion-dependent keratin polymerization occurs in forming lamellipodia and that transport of newly formed keratin particles is mediated by actin filaments until network integration. This mechanism allows extension of the KF network toward the leading edge in migrating cells and may be of relevance for tissue development and regeneration.

The keratin-filament cycle of assembly and disassembly

“…The identification of diffusing keratins shows that active transport machinery is not needed to deliver disassembled keratins to the cell periphery for KF reassembly. This is also supported by the finding that KFP formation persists, although at reduced levels, in the presence of actin filament and microtubule disruptors (Kolsch et al, 2009;Woll et al, 2005). The soluble pool is also available for lateral exchange into existing filaments (Miller et al, 1991), which might explain the slight recovery of bleached filaments in our recordings (e.g.…”

“…It was observed by time-lapse fluorescence recording of cell lines producing fluorescent epithelial KFs that KF formation occurs preferentially at free cell edges and adjoining cell borders of stationary cells (Kolsch et al, 2009;. After scratch wounding, abundant KF precursors (KFPs) were seen at the leading edge of approaching cells, thereby extending the IF cytoskeleton towards the gap ( Fig.…”

“…The actin retrograde flow and ruffling activity (Small and Resch, 2005) might provide the driving force for centripetal KF movement (Kolsch et al, 2009;Windoffer et al, 2006;Windoffer and Leube, 2001;Woll et al, 2005). Microtubules might also be needed, especially for the subsequent inward motion of the entire network (for a review, see Helfand et al, 2004).…”

“…Newly formed KFPs are continuously transported toward the nucleus, relying primarily on intact actin filaments (Kolsch et al, 2009;Woll et al, 2005). To further track KFs after integration into the peripheral network, image-processing algorithms were developed and applied to motility analyses.…”

Regulation of keratin expression by retinoids

“…[21][22][23][24][25][26] In normal human epidermis, heterodimers of keratin 5 (K5) and K14 form the cytoskeleton of undifferentiated cells in the basal layer, which is replaced in suprabasal cells by K1 and K10, accompanied by K2 in the review review and KRT17. [28][29][30] Upon differentiation of keratinocytes, e.g., by raising the calcium level with or without the addition of growth factors (EGF and fibroblast growth factor-10) or adding the EGF-receptor inhibitor PD153035, expression of the differentiation-related keratins K1 and K10 are induced in a subpopulation of cells.…”