Identification of Novel Principles of Keratin Filament Network Turnover in Living Cells

Abstract

It is generally assumed that turnover of the keratin filament system occurs by exchange of subunits along its entire length throughout the cytoplasm. We now present evidence that a circumscribed submembranous compartment is actually the main site for network replenishment. This conclusion is based on the following observations in living cells synthesizing fluorescent keratin polypeptides: 1) Small keratin granules originate in close proximity to the plasma membrane and move toward the cell center in a continuous motion while elongating into flexible rod-like fragments that fuse with each other and integrate into the peripheral KF network. 2) Recurrence of fluorescence after photobleaching is first seen in the cell periphery where keratin filaments are born that translocate subsequently as part of the network toward the cell center. 3) Partial keratin network reformation after orthovanadate-induced disruption is restricted to a distinct peripheral zone in which either keratin granules or keratin filaments are transiently formed. These findings extend earlier investigations of mitotic cells in which de novo keratin network formation was shown to originate from the cell cortex. Taken together, our results demonstrate that the keratin filament system is not homogeneous but is organized into temporally and spatially distinct subdomains. Furthermore, the cortical localization of the regulatory cues for keratin filament turnover provides an ideal way to adjust the epithelial cytoskeleton to dynamic cellular processes.

Simulating the formation of keratin filament networks by a piecewise-deterministic Markov process

“…High network connectivity seems to be favored by rapid elongation of the filaments once they have been initiated. Rapid filament elongation has also been observed in vivo by Windoffer et al (2004) and may thus be a key factor for the high degree of connectivity found in image data of keratin networks. This process, however, might be restricted by the diffusion-limited supply with soluble keratin oligomers.…”

“…Thus, the model has been designed for small observation windows. In our previous studies, we investigated two-dimensional electron microscopy images of keratin networks taken from peripheral cytoplasmatic compartments (Beil et al, 2005 and2006) which is pivotal for keratin network dynamics (Windoffer et al, 2004). These compartments are very thin and contain mostly a single layer of keratin filaments.…”

“…1A). Windoffer et al (2004) report the observation of nucleation phenomena by means of fluorescence microscopy.…”

“…1D). Merging has been observed in vivo by Windoffer et al (2004). In electron microscopy images of keratin networks a certain fraction of the inter-filament connections are of degree 4.…”

“…Filament initiation (event A 1 ) includes nucleation as well as lateral annealing. Observations by Windoffer et al (2004) suggest that the initiation of new filaments is preceded by the formation of small keratin clusters. Therefore, this event is assumed to require high levels of soluble keratin concentration.…”