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.
(c) 2009 Wiley-Liss, Inc.