Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM)

Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several µm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 µm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The ‘patchy’ structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments

Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16.

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2-/- mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this 'stress' keratin is regulated

The Intermediate Filament Network in Cultured Human Keratinocytes Is Remarkably Extensible and Resilient

The prevailing model of the mechanical function of intermediate filaments in cells assumes that these 10 nm diameter filaments make up networks that behave as entropic gels, with individual intermediate filaments never experiencing direct loading in tension. However, recent work has shown that single intermediate filaments and bundles are remarkably extensible and elastic in vitro, and therefore well-suited to bearing tensional loads. Here we tested the hypothesis that the intermediate filament network in keratinocytes is extensible and elastic as predicted by the available in vitro data. To do this, we monitored the morphology of fluorescently-tagged intermediate filament networks in cultured human keratinocytes as they were subjected to uniaxial cell strains as high as 133%. We found that keratinocytes not only survived these high strains, but their intermediate filament networks sustained only minor damage at cell strains as high as 100%. Electron microscopy of stretched cells suggests that intermediate filaments are straightened at high cell strains, and therefore likely to be loaded in tension. Furthermore, the buckling behavior of intermediate filament bundles in cells after stretching is consistent with the emerging view that intermediate filaments are far less stiff than the two other major cytoskeletal components F-actin and microtubules. These insights into the mechanical behavior of keratinocytes and the cytokeratin network provide important baseline information for current attempts to understand the biophysical basis of genetic diseases caused by mutations in intermediate filament genes

Gene Expression Changes Associated with the Airway Wall Response to Injury

Understanding the way in which the airway heals in response to injury is fundamental to dissecting the mechanisms underlying airway disease pathology. As only limited data is available in relation to the in vivo characterisation of the molecular features of repair in the airway we sought to characterise the dynamic changes in gene expression that are associated with the early response to physical injury in the airway wall.We profiled gene expression changes in the airway wall using a large animal model of physical injury comprising bronchial brush biopsy in anaesthetised sheep. The experimental design featured sequential studies in the same animals over the course of a week and yielded data relating to the response at 6 hours, and 1, 3 and 7 days after injury. Notable features of the transcriptional response included the early and sustained preponderance of down-regulated genes associated with angiogenesis and immune cell activation, selection and differentiation. Later features of the response included the up-regulation of cell cycle genes at d1 and d3, and the latter pronounced up-regulation of extracellular matrix-related genes at d3 and d7.It is possible to follow the airway wall response to physical injury in the same animal over the course of time. Transcriptional changes featured coordinate expression of functionally related genes in a reproducible manner both within and between animals. This characterisation will provide a foundation against which to assess the perturbations that accompany airway disease pathologies of comparative relevance

Non-Coding Keratin Variants Associate with Liver Fibrosis Progression in Patients with Hemochromatosis

Background: Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with endstage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods: The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCRamplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previouslygenerated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results: We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. I

Silencing cytokeratin 18 gene inhibits intracellular replication of Trypanosoma cruzi in HeLa cells but not binding and invasion of trypanosomes

<p>Abstract</p> <p>Background</p> <p>As an obligatory intracellular parasite, <it>Trypanosoma cruzi</it>, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18) is among the host molecules that have been suggested as a mediator of important events during <it>T. cruzi</it>-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi)-mediated down regulation of the CK18 gene could interfere with the parasite life cycle <it>in vitro</it>. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence.</p> <p>Results</p> <p>CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different <it>T. cruzi </it>strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18.</p> <p>Conclusion</p> <p>The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of <it>T. cruzi </it>in HeLa cells, but not trypanosome binding and invasion.</p

Characterization of In Vivo Keratin 19 Phosphorylation on Tyrosine-391

Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported.In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the 'tail' domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively.Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic

Keratin 8 expression in colon cancer associates with low faecal butyrate levels

<p>Abstract</p> <p>Background</p> <p>Butyrate has been implicated in the mechanistic basis of the prevention of colorectal cancer by dietary fibre. Numerous in vitro studies have shown that butyrate regulates cell cycle and cell death. More recently we have shown that butyrate also regulates the integrity of the intermediate filament (IF) cytoskeleton <it>in vitro</it>. These and other data suggest a link between the role of diet and the implication of a central role for the keratin 8 (K8) as guardian of the colorectal epithelium.</p> <p>Methods</p> <p>In this cross-sectional study possible links between butyrate levels, field effects and keratin expression in cancer were addressed directly by analysing how levels of expression of the IF protein K8 in tumours, in adjacent fields and at a distant landmark site may be affected by the level of butyrate in the colon microenvironment. An immunohistochemical scoring protocol for K8 was developed and applied to samples, findings were further tested by immunoblotting.</p> <p>Results</p> <p>Levels of K8 in colorectal tumours are lower in subjects with higher levels of faecal butyrate. Immunoblotting supported this finding.Although there were no significant relationships with butyrate on the non-tumour tissues, there was a consistent trend in all measures of extent or intensity of staining towards a reduction in expression with elevated butyrate, consistent with the inverse association in tumours.</p> <p>Conclusions</p> <p>The data suggest that butyrate may associate with down-regulation of the expression of K8 in the cancerized colon. If further validated these findings may suggest the chemopreventive value of butyrate is limited to early stage carcinogenesis as low K8 expression is associated with a poor prognosis.</p

Protein disulphide isomerase-mediated grafting of cysteine-containing peptides onto over-bleached hair

The ability of Protein disulphide isomerase (PDI) to promote the grafting of two cysteine-containing peptides onto hair was investigated in order to develop an alternative treatment for over-bleached hair. The studied peptides were designed based on human keratin and human lung surfactant proteins and were linked to a fluorescent dye to facilitate visualisation of the grafting process and to assess hair penetration. The ability of the peptides to restore mechanical and thermal properties lost by repeated bleaching treatments was also studied. After eight bleaching treatments, hair samples displayed 42% less mechanical resistance, coupled with a decrease in α-helix denaturation enthalpies and temperatures. Hair surface damage following bleaching was visualized by scanning electron microscopy. Addition of PDI to the treatment formulations promoted peptide attachment to the hair via disulphide bonds, facilitating their penetration into the hair cortex, as observed by fluorescence microscopy. The proposed peptide treatment resulted in an increase in α-helix denaturation enthalpy in over-bleached hair, as well as an increase in both Young's modulus and tensile strength. Thus, mechanical and thermal properties were improved after the peptide treatment in the presence of PDI; suggesting that the formulations presented in this work are promising candidates for hair-care applications

Genome-Wide Expression Profiling of Five Mouse Models Identifies Similarities and Differences with Human Psoriasis

Development of a suitable mouse model would facilitate the investigation of pathomechanisms underlying human psoriasis and would also assist in development of therapeutic treatments