Order and disorder in intermediate filament proteins

AbstractIntermediate filaments (IFs), important components of the cytoskeleton, provide a versatile, tunable network of self-assembled proteins. IF proteins contain three distinct domains: an α-helical structured rod domain, flanked by intrinsically disordered head and tail domains. Recent studies demonstrated the functional importance of the disordered domains, which differ in length and amino-acid sequence among the 70 different human IF genes. Here, we investigate the biophysical properties of the disordered domains, and review recent findings on the interactions between them. Our analysis highlights key components governing IF functional roles in the cytoskeleton, where the intrinsically disordered domains dictate protein–protein interactions, supramolecular assembly, and macro-scale order

Phosphorylation-Induced Mechanical Regulation of Intrinsically Disordered Neurofilament Proteins

The biological function of protein assemblies has been conventionally equated with a unique three-dimensional protein structure and protein-specific interactions. However, in the past 20 years it has been found that some assemblies contain long flexible regions that adopt multiple structural conformations. These include neurofilament proteins that constitute the stress-responsive supportive network of neurons. Herein, we show that the macroscopic properties of neurofilament networks are tuned by enzymatic regulation of the charge found on the flexible protein regions. The results reveal an enzymatic (phosphorylation) regulation of macroscopic properties such as orientation, stress response, and expansion in flexible protein assemblies. Using a model that explains the attractive electrostatic interactions induced by enzymatically added charges, we demonstrate that phosphorylation regulation is far richer and versatile than previously considered

Probing the Interactions of Intrinsically Disordered Proteins Using Nanoparticle Tags

The structural plasticity of intrinsically disordered proteins serves as a rich area for scientific inquiry. Such proteins lack a fix three-dimensional structure but can interact with multiple partners through numerous weak bonds. Nevertheless, this intrinsic plasticity possesses a challenging hurdle in their characterization. We underpin the intermolecular interactions between intrinsically disordered neurofilaments in various hydrated conditions, using grafted gold nanoparticle (NP) tags. Beyond its biological significance, this approach can be applied to modify the surface interaction of NPs for the creation of future tunable “smart” hybrid biomaterials

Met Kinetic Signature Derived from the Response to HGF/SF in a Cellular Model Predicts Breast Cancer Patient Survival

To determine the signaling pathways leading from Met activation to metastasis and poor prognosis, we measured the kinetic gene alterations in breast cancer cell lines in response to HGF/SF. Using a network inference tool we analyzed the putative protein-protein interaction pathways leading from Met to these genes and studied their specificity to Met and prognostic potential. We identified a Met kinetic signature consisting of 131 genes. The signature correlates with Met activation and with response to anti-Met therapy (p,0.005) in in-vitro models. It also identifies breast cancer patients who are at high risk to develop an aggressive disease in six large published breast cancer patient cohorts (p,0.01, N.1000). Moreover, we have identified novel putative Met pathways, which correlate with Met activity and patient prognosis. This signature may facilitate personalized therapy by identifying patients who will respond to anti-Met therapy. Moreover, thi

The role of Vimentin in Regulating Cell Invasive Migration in Dense Cultures of Breast Carcinoma Cells

Cell migration and mechanics are tightly regulated by the integrated activities of the various cytoskeletal networks. In cancer cells, cytoskeletal modulations have been implicated in the loss of tissue integrity and acquisition of an invasive phenotype. In epithelial cancers, for example, increased expression of the cytoskeletal filament protein vimentin correlates with metastatic potential. Nonetheless, the exact mechanism whereby vimentin affects cell motility remains poorly understood. In this study, we measured the effects of vimentin expression on the mechano-elastic and migratory properties of the highly invasive breast carcinoma cell line MDA231. We demonstrate here that vimentin stiffens cells and enhances cell migration in dense cultures, but exerts little or no effect on the migration of sparsely plated cells. These results suggest that cell–cell interactions play a key role in regulating cell migration, and coordinating cell movement in dense cultures. Our findings pave the way toward understanding the relationship between cell migration and mechanics in a biologically relevant context

Met Kinetic Signature correlation with clinical and histopathological data.

<p>Met Kinetic Signature correlation with clinical and histopathological data.</p

Molecular analysis of Met kinetic signature- mRNA and protein levels of selected genes in high and low Met expressing cells.

<p>(A) Total cellular RNA, was isolated from low (MCF7) and high Met (MDA231) cell cultures and mRNA expression of Met, Survivin, Pbk, Cyclin E1 and Ki67 was evaluated by quantitative real time PCR and compared mRNA levels of the housekeeping GAPDH gene. The primers used for the quantification of gene expression are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045969#pone.0045969.s011" target="_blank">Table S2</a>. A gray box denotes MCF7 cell line samples and a black box denotes MDA231 cell line samples (B) Samples from low (MCF7) and high Met (MDA231) cells were subjected to western blot (WB) analysis, before and 15 min and 60 min after treatment with HGF/SF, using antibodies against Met and activated Met (p-Met) and (C) antibodies against ERK K-23, p-ERK E-4, E-Cadherin, Survivin and Actin C4. (D, E) Subcellular localization of survivin in fluorescence (IF) analysis of Low (MCF7) and high Met (MDA231) cells after treatment with HGF/SF at 0 min, 10 min, 30 min and 24 h. The cells were Immunostained using anti-Survivin antibody. Immunofluorescence was examined using a 510 Meta Zeiss confocal laser scanning microscope (CLSM). Survivin quantification was performed on at least five confocal images per slide. Cell outline was defined based on Nomarski images; nuclei were defined based on the DAPI staining. Average pixel intensity was calculated separately for the nucleus and cytoplasm areas. (F) IF analysis of temporal kinetics of Survivin protein expression following treatment with HGF/SF.</p

Met signature segmentation of cell line model and human breast cancer patients’ data sets.

<p>(A) Cells from six human breast cancer cell lines (MDA231, Hs578T, BT549, MCF10, MCF7 and T47D) were incubated with purified HGF/SF labeled with biotin by a protein biotinylation kit and allowed to bind for 30 min. Cells were then fixed with 4% Paraformaldehyde, permeablized, and stained with Streptavidin-coupled QDot585. Fluorescence levels calculated by image analysis using MICA image analysis software, p<0.0001. (B) Met canonical pathway score calculated by measuring the average mRNA levels of all Met canonical pathway genes (after normalization to average = 0, stdev = 1 per-gene) in high-Met (MDA231, Hs578T and BT549) as compared to the low-Met (MCF10, MCF7 and T47D) samples, p<0.0001. A gray box denotes high Met cell line samples and a black box denotes low Met cell line samples. (C) Hierarchical clustering division of breast cancer cell lines samples using Met kinetic signature genes.</p