Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases

Abstract

The cytokeratin filament network is intrinsically dynamic, continuously exchanging subunits over its entire surface, while conferring structural stability on epithelial cells. However, it is not known how cytokeratin filaments are remodeled in situations where the network is temporarily and spatially restricted. Using the tyrosine phosphatase inhibitor orthovanadate we observed rapid and reversible restructuring in living cells, which may provide the basis for such dynamics. By examining cells stably expressing fluorescent cytokeratin chimeras, we found that cytokeratin filaments were broken down and then formed into granular aggregates within a few minutes of orthovanadate addition. After drug removal, gradual reincorporation of granules into the filament network was observed for aggregates that were either part of residual filaments or stayed in close apposition to remaining filaments. Even when cytokeratin filaments were no longer detectable, granules with low mobility were still able to reestablish a cytokeratin filament network. This process took less than 30 minutes and occurred at multiple foci throughout the cytoplasm without apparent correlation to alterations in the actin- and tubulin-based systems. Interestingly, the short-lived and rather small orthovanadate-induced cytokeratin granules contained the cytoskeletal crosslinker plectin but lacked the cytokeratin-solubilising 14-3-3 proteins. By contrast, the long-lived and larger cytokeratin aggregates generated after treatment with the serine/threonine phosphatase inhibitor okadaic acid were negative for plectin but positive for 14-3-3 proteins. Taken together, our observations in living orthovanadate-treated interphase cells revealed modes of cytokeratin remodeling that qualify as basic mechanisms capable of rapidly adapting the cytokeratin filament cytoskeleton to specific requirements.

14-3-3σ stabilizes a complex of soluble actin and intermediate filament to enable breast tumor invasion

“…5E). Other 14-3-3 family members were shown previously to regulate intermediate filament architecture by functioning as K8/K18 solubility cofactors in a PKCζ-dependent mechanism (40)(41)(42). In addition, a number of studies have highlighted different mechanisms by which 14-3-3 family members regulate actin dynamics either through stabilizing cofilin phosphorylation or by inhibiting signals through the AKT-RhoA pathway (12,(43)(44)(45).…”

“…It is known that cytokeratins are rapidly depolymerized in the presence of phosphatase inhibitors such as the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, indicating that intermediate filament homeostasis is regulated by phosphorylation (40). Furthermore, it was shown previously that protein kinase Cζ (PKCζ) phosphorylates K18 at a 14-3-3-binding site, leading to K8/K18 solubilization through interaction with 14-3-3ζ (41,42).…”

Investigating Cellular Nanoscale with X-rays

“…In cells, keratin proteins form bundles and networks. The mechanical and structural properties of keratin bundles have been studied by microscopy techniques [103,117,[183][184][185][186][187][188][189][190][191], but the internal structure could not be accessed. One way to access the internal architecture of these bundles is by the use of X-rays.…”

“…We used SK8K18 cells [184,185], stably transfected with DNA encoding for fluorescent keratin hybrids (HK8-CFP, HK18-YFP) from SW13 cell line (human adrenal cortex carcinoma). The cells were grown in culture Petri dishes using high glucose (4.5 g/L) Dulbecco's modified eagle medium (DMEM), with 10% (v/v) fetal calf serum (FCS), 100 units/mL penicillin, and 0.1 mg/mL streptomycin at 37 • C in a water-saturated atmosphere with 5% CO 2 .…”

“…SK8K18 cells [59,76,184,185] have been fixed with three widely used fixatives [241][242][243][244]: i) formaldehyde (F), consisting of a solution of 37% formaldehyde supplemented with 10% of methanol as stabiliser (in order to avoid the polymerisation of the formaldehyde into parafromaldehyde chains) and diluted 10× in PBS (phosphate-buffered saline), ii) paraformaldehyde (PFA), which is a polymerisation product of formaldehyde, consisting of 8 to 100 units and iii) methanol (MeOH). The experiments were performed in September 2013 at the P10 beamline described in section 2.6.1 with the following specificities:…”