Edited by Norma AllewellIntermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They are not only responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil-forming proteins exhibiting a characteristic central ␣-helical domain ("rod"). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also, a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend these static data and to investigate the dynamic properties of the full-length proteins in solution during the various assembly steps, we analyzed the patterns of hydrogen-deuterium exchange in vimentin and in four variants carrying point mutations in the IF consensus motifs present at either end of the ␣-helical rod that cause an assembly arrest at the unit-length filament (ULF) stage. The results yielded unique insights into the structural properties of subdomains within the full-length vimentin, in particular in regions of contact in ␣-helical and linker segments that stabilize different oligomeric forms such as tetramers, ULFs, and mature filaments. Moreover, hydrogen-deuterium exchange analysis of the point-mutated variants directly demonstrated the active role of the IF consensus motifs in the oligomerization mechanism of tetramers during ULF formation. Ultimately, using molecular dynamics simulation procedures, we provide a structural model for the subdomain-mediated tetramer/tetramer interaction via "crosscoiling" as the first step of the assembly process. Intermediate filament (IF)3 proteins form highly resilient filaments that are markedly resistant to mechanical stress. Mediated by prominent cytolinker proteins of the plakin family and motor proteins, they integrate actin filaments and microtubules to establish a functional cytoskeleton in metazoan cells and promote optimal tissue function (1). Apart from their basic mechanical function in maintaining cell flexibility, they are also involved in multiple cellular activities that range from cell division and motility to the topological organization of transmembrane channels (2, 3). Because of these multifunctional properties of IF proteins, mutations in IF-encoding genes cause almost 100 different inherited diseases in humans (4, 5). One of the most studied IF proteins, vimentin, forms IFs typical for mesenchymal cells, including endothelial cells, lymphocytes, and the eye lens epithelium (6). Of note, vimentin has long been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-mesenchymal transition, and the metastatic spread of cancer (7), making this protein an attractive potential target for cancer diagnosis and therapy (8). Therefore, to develop mechanistic insight into the beh...

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Keratins are intermediate filament (IF) proteins that form complex filament systems in epithelial cells, thus serving as scaffolding elements and mechanical stress absorbers. The building blocks of keratin IFs are parallel coiled-coil dimers of two distinct sequence-related proteins distinguished as type I and type II keratins. To gain more insight into their structural dynamics, we resorted to hydrogen-deuterium exchange mass spectrometry of keratins K8 and K18, which are characteristic for simple epithelial cells. Using this powerful technique not employed with IFs before, we mapped patterns of protected versus unprotected regions in keratin complexes at various assembly levels. In particular, we localized protein segments exhibiting different hydrogen exchange patterns in tetramers versus filaments. We observed a general pattern of precisely positioned regions of stability intertwining with flexible regions, mostly represented by the non-α-helical segments. Notably, some regions within the coiled-coil domains are significantly more dynamic than others, while the IF-consensus motifs at the end domains of the central α-helical "rod" segment, which mediate the "head-to-tail" dimer-dimer interaction in the filament elongation process, become distinctly more protected upon formation of filaments. Moreover, to gain more insight into the dynamics of the individual keratins, we investigated the properties of homomeric preparations of K8 and K18. The physiological importance of keratins without a partner is encountered in both pathological and experimental situations when one of the two species is present in robust excess or completely absent, such as in gene-targeted mice.

Genetic and acquired loss-of-function defect of the cystic fibrosis transmembrane conductance regulator (CFTR) compromise airway surface liquid homeostasis and mucociliary clearance (MCC), culminating in recurrent lung inflammation/infection. While chronic cigarette smoke (CS), CS extract (CSE; water-soluble compounds) and CS condensate (CSC; particulate, organic fraction) exposure inhibit CFTR activity at transcriptional, biochemical, and functional levels, the acute impact of CSC remains incompletely understood. We report that CSC transiently activates CFTR chloride secretion in airway epithelia. The comparable CFTR phospho-occupancy after CSC- and forskolin-exposure, determined by affinity-enriched tandem mass spectrometry and pharmacology, suggest that localised cAMP-dependent protein kinase (PKA) stimulation by CSC causes the channel opening. Due to the inhibition of the MRP4/ABCC4, a cAMP-exporter confined to the CFTR macromolecular signalling-complex, PKA activation is accomplished by the subcompartmentalised elevation of cytosolic cAMP. In line, MRP4 inhibition results in CFTR activation and phospho-occupancy similar to that by forskolin. In contrast, acute CSC exposure reversibly inhibits the phosphorylated CFTR both in vivo and in phospholipid bilayers, without altering its cell surface density and phospho-occupancy. We propose that components of CSC elicit both a transient protective CFTR activation, as well as subsequent channel block in airway epithelia, contributing to the subacute MCC defect in acquired CF lung diseases.