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Intermediate filaments (IFs) are integral components of the cytoskeleton which provide cells with tissue-specific mechanical properties and are involved in a plethora of cellular processes. Unfortunately, due to their intricate architecture, the 3D structure of the complete molecule of IFs has remained unresolved. Even though most of the rod domain structure has been revealed by means of crystallographic analyses, the flanked head and tail domains are still mostly unknown. Only recently have studies shed light on head or tail domains of IFs, revealing certainsecondary structures and conformational changes during IF assembly. Thus, a deeper understanding of their structure could provide insights into their function.

Intermediate filaments (IFs), being traditionally the least studied component of the cytoskeleton, have begun to receive more attention in recent years. IFs are found in different cell types and are specific to them. Accumulated data have shifted the paradigm about the role of IFs as structures that merely provide mechanical strength to the cell. In addition to this role, IFs have been shown to participate in maintaining cell shape and strengthening cell adhesion. The data have also been obtained that point out to the role of IFs in a number of other biological processes, including organization of microtubules and microfilaments, regulation of nuclear structure and activity, cell cycle control, and regulation of signal transduction pathways. They are also actively involved in the regulation of several aspects of intracellular transport. Among the intermediate filament proteins, vimentin is of particular interest for researchers. Vimentin has been shown to be associated with a range of diseases, including cancer, cataracts, Crohn's disease, rheumatoid arthritis, and HIV. In this review, we focus almost exclusively on vimentin and the currently known functions of vimentin intermediate filaments (VIFs). This is due to the structural features of vimentin, biological functions of its domains, and its involvement in the regulation of a wide range of basic cellular functions, and its role in the development of human diseases. Particular attention in the review will be paid to comparing the role of VIFs with the role of intermediate filaments consisting of other proteins in cell physiology.

After the initial discovery of intermediate filament (IF)-forming proteins in 1968, a decade would elapse before they were revealed to comprise a diverse group of proteins which undergo tissue-, developmental stage-, differentiation-, and context-dependent regulation. Our appreciation for just how large (n = 70), conserved, complex, and dynamic IF genes and proteins are became even sharper upon completion of the human genome project. While there has been extraordinary progress in understanding the multimodal roles of IFs in cells and tissues, even revealing them as direct causative agents in a broad array of human genetic disorders, the link between individual IFs and cell differentiation has remained elusive. Here, we review evidence that demonstrates a role for IFs in lineage determination, cell differentiation, and tissue homeostasis. A major theme in this review is the function of IFs as sensors and transducers of mechanical forces, intersecting microenvironmental cues and fundamental processes through cellular redox balance., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

DNA methylation contributes to malignant transformation, but little is known about how the methylation drives colorectal cancer evolution at the early stages. Here we identify aberrant INA (α-internexin) gene methylation in colon adenoma and adenocarcinoma by filtering data obtained from a genome-wide screen of methylated genes. The gene encoding INA, a type IV intermediate filament, was frequently hypermethylated in CpG islands located in the promoter region. This hypermethylation preferentially occurred in large tumors and was a prognostic marker for poor overall survival in patients with colorectal cancer. This type of epigenetic alteration silenced INA expression in both adenoma and adenocarcinoma tissues. Gene silencing of INA in colorectal cancer cells increased cell proliferation, migration, and invasion. Restored INA expression blocked migration and invasion in vitro and reduced lung metastasis in vivo . Mechanistically, INA directly inhibited microtubule polymerization in vitro and decreased intracellular microtubule plus-end assembly rates. A peptide array screen surveying the tubulin-binding sites in INA identified a tubulin-binding motif located in the N-terminal head domain that plays a tumor-suppressive role by binding to unpolymerized tubulins and impeding microtubule polymerization. Thus, epigenetic inactivation of INA is an intermediate filament reorganization event that is essential to accelerate microtubule polymerization in the early stages of colorectal cancer. SIGNIFICANCE: This work provides insight into the epigenetic inactivation of INA, a novel identified tumor suppressor, which increases microtubule polymerization during colorectal cancer progression., (©2020 American Association for Cancer Research.)

Deimination (or citrullination) is a post-translational modification catalyzed by a calcium-dependent enzyme family of five peptidylarginine deiminases (PADs). Deimination is involved in physiological processes (cell differentiation, embryogenesis, innate and adaptive immunity, etc.) and in autoimmune diseases (rheumatoid arthritis, multiple sclerosis and lupus), cancers and neurodegenerative diseases. Intermediate filaments (IF) and associated proteins (IFAP) are major substrates of PADs. Here, we focus on the effects of deimination on the polymerization and solubility properties of IF proteins and on the proteolysis and cross-linking of IFAP, to finally expose some features of interest and some limitations of citrullinomes.

The native hagfish slime threads, which are made up of two intermediate filament (IF)-like proteins, exhibit mechanical properties comparable to dragline spider silk fiber, the toughest fiber in nature. However, unlike silk, the design of artificial IF-protein-based fibers has been rarely studied, possibly because the unique hierarchical organization of the keratin-like proteins within these threads is challenging to mimic, and consequently, extraordinary fiber mechanics has not been shown in slime threads from recombinant IF-protein-based system. Here, we have reported the synthesis and properties of recombinant type V IF-protein, based on the Caenorhabditis elegans (Ce) lamin gene. The protein was solubilized and wet-spun into aqueous solutions to prepare Ce-lamin fibers by varying injection flow rates and Ca +2 ion concentrations in the coagulation buffer. At specific set of conditions, Ce-lamin fibers demonstrated remarkable toughness and stiffness, comparable to hagfish slime threads and natural dragline spider silk. Transmission electron microscopy analysis showed that paracrystals were the main nanometric structure within the fibers. This study demonstrates that outstanding mechanical properties can be achieved with recombinant IF-proteins through self-organization. Thus, these results have broadened the pool of fibrous proteins that can be used in functional materials for a diverse range of applications., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent on either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Drosophila Tm1-I/C IFs functions by forming labile cross-β interactions. These interactions are seen in polymers made from the tail domain alone, as well as in assembled IFs formed by the intact Tm1-I/C protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Terbinafine (Tbf) is a well-established anti-fungal agent used for management of a variety of dermal conditions including ringworm and athlete's foot. Both the biochemical mechanism of Tbf fungicidal action (based on squalene epoxidase inhibition) and the target region for Tbf in vivo (the stratum corneum (SC)) are well determined. However, the biochemical and pharmacokinetic approaches used to evaluate Tbf biochemistry provide no biophysical information about molecular level physical changes in the SC upon Tbf binding. Such information is necessary for improved drug and formulation design. IR spectroscopic methods were used to evaluate the effects of Tbf on keratin structure in environments commonly used in pharmaceutics to mimic those in vivo. The Amide I and II spectral regions (1500-1700 cm -1 ) provided an effective means to monitor keratin secondary structure changes, while a Tbf spectral feature near 775 cm -1 provides a measure of relative Tbf levels in skin. Interaction of Tbf with the SC induced substantial β-sheet formation in the keratin, an effect which was partially reversed both by ethanol washing and by exposure to high relative humidity. The irreversibility suggests the presence of a Tbf reservoir (consistent with kinetic studies), permitting the drug to be released in a controlled manner into the surrounding tissue., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Despite the remarkable complexity of the individual neuron and of neuronal circuits, it has been clear for quite a while that, in order to understand the functioning of the brain, the contribution of other cell types in the brain have to be accounted for. Among glial cells, astrocytes have multiple roles in orchestrating neuronal functions. Their communication with neurons by exchanging signaling molecules and removing molecules from extracellular space takes place at several levels and is governed by different cellular processes, supported by multiple cellular structures, including the cytoskeleton. Intermediate filaments in astrocytes are emerging as important integrators of cellular processes. Astrocytes express five types of intermediate filaments: glial fibrillary acidic protein (GFAP); vimentin; nestin; synemin; lamins. Variability, interactions with different cellular structures and the particular roles of individual intermediate filaments in astrocytes have been studied extensively in the case of GFAP and vimentin, but far less attention has been given to nestin, synemin and lamins. Similarly, the interplay between different types of cytoskeleton and the interaction between the cytoskeleton and membranous structures, which is mediated by cytolinker proteins, are understudied in astrocytes. The present review summarizes the basic properties of astrocytic intermediate filaments and of other cytoskeletal macromolecules, such as cytolinker proteins, and describes the current knowledge of their roles in normal physiological and pathological conditions.

Atopic dermatitis (AD) is a chronic inflammatory disease that affects approximately 2-5% of adults worldwide. The pathogenesis of AD continues to be a well-debated point of conjecture, with numerous hypotheses having been proposed. AD conditions are associated with increased populations of Staphylococcus aureus and reduced skin lipids. In this study, we evaluate the ability of S. aureus to permeate across human stratum corneum (SC) exhibiting both normal and depleted lipid conditions consistent with AD. This permeation would enable bacteria to interact with underlying viable epidermal cells, which could serve as a trigger for inflammation and disease onset. Our results indicate that permeation of S. aureus through SC exhibiting normal lipid conditions is not statistically significant. However, bacteria can readily permeate through lipid depleted tissue over a 9-d period. These findings suggest that S. aureus may potentially act as the mechanistic cause, rather than merely the result of AD., Abbreviations: AD: Atopic dermatitis; SC: Stratum Corneum; AMP: Antimicrobial peptide; DIW: Deionized water; PDMS: Polydimethylsiloxane; GFP: Green fluorescent protein; BHI: Brain heart infusion medium.

X-band (9.4 GHz) electron paramagnetic resonance (EPR) and electron paramagnetic resonance imaging (EPRI) were used for elucidating structural aspects of the stratum corneum (SC). We found that psoriasis vulgaris (PV)-SC has a less-ordered structure than that of the control SC, indicating the abnormal architecture of PV-SC. Different spectra were observed for the PV-SC. The three-line spectral pattern suggests that the 5-doxylstearic acid (5-DSA) is mobile or less rigid in the SC. The simulated order parameter (S 0 ) value obtained for 5-DSA in the SC was approximately 0.20. The statistical analysis suggests that the value of the PV-SC is significantly smaller than that of the control (p < 0.01). Thus, we suggest that this EPR assay is of great use for evaluating SC function. In addition, the EPRI of various SC samples provides a useful image concerning SC status. A strong red image was observed for the PV skin. No red lesion region was observed in the control. The EPR images differentiated various sizes and number distribution concerning the disordered states in the SC.

Analysis of fatty acid contents and pharmacological properties of Pliek U oil was performed. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC-MS), and pharmacological properties based on its potential on filament-aggregating protein (filaggrin) were studied with bioinformatics approach by the reverse docking technique using palmitic acid as a control compound. Two Pliek U extracts, namely, Pliek U oil (PUO) and ethanolic Pliek U oil extract (EPUOE), were prepared. The GC-MS results revealed that lauric acid, myristic acid, palmitic acid, and oleic acid are the predominant fatty acids, with lauric acid being the abundant one in all Pliek U oil extracts. The reverse docking technique results showed that oleic acid had the most stable interaction to filaggrin with the lowest binding affinity (-6.1 kcal/mol). Oleic acid and palmitic acid have one same side binding to filaggrin on amino acid LEU D75. These findings indicated that oleic acid has the best potential to be used as a drug candidate in atopic dermatitis treatment., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2019 Nanda Earlia et al.)

Lamin A is a nuclear intermediate filament protein critical for nuclear architecture and mechanics and mutated in a wide range of human diseases. Yet little is known about the molecular architecture of lamins and mechanisms of their assembly. Here we use SILAC cross-linking mass spectrometry to determine interactions within lamin dimers and between dimers in higher-order polymers. We find evidence for a compression mechanism where coiled coils in the lamin A rod can slide onto each other to contract rod length, likely driven by a wide range of electrostatic interactions with the flexible linkers between coiled coils. Similar interactions occur with unstructured regions flanking the rod domain during oligomeric assembly. Mutations linked to human disease block these interactions, suggesting that this spring-like contraction can explain in part the dynamic mechanical stretch and flexibility properties of the lamin polymer and other intermediate filament networks.

To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 Å resolution. It revealed biochemical determinants for the A 11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227 K1 , Y230 K1 , F231 K1 , and F234 K1 ), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314 K1 and L318 K1 ). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314A K1 , but not L318A K1 , abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 Å structure of K1/10-1B containing a S233L K1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1 S233L /K10-1B in solution without affecting secondary structure. The K1 S233L /K10-1B octamer structure revealed S233L K1 causes aberrant hydrophobic interactions between 1B tetramers., (© 2019 The Authors.)

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress, which induce extensive reorganization of the vimentin cytoskeletal network. Here, we have investigated the mechanisms underlying these effects. In vitro, electrophilic lipids, including 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) and 4-hydroxynonenal (HNE), directly bind to vimentin, whereas the oxidant diamide induces disulfide bond formation. Mutation of the single vimentin cysteine residue (Cys328) blunts disulfide formation and reduces lipoxidation by 15d-PGJ 2 , but not HNE. Preincubation with these agents differentially hinders NaCl-induced filament formation by wild-type vimentin, with effects ranging from delayed elongation and increased filament diameter to severe impairment of assembly or aggregation. Conversely, the morphology of vimentin Cys328Ser filaments is mildly or not affected. Interestingly, preformed vimentin filaments are more resistant to electrophile-induced disruption, although chemical modification is not diminished, showing that vimentin (lip)oxidation prior to assembly is more deleterious. In cells, electrophiles, particularly diamide, induce a fast and drastic disruption of existing filaments, which requires the presence of Cys328. As the cellular vimentin network is under continuous remodeling, we hypothesized that vimentin exchange on filaments would be necessary for diamide-induced disruption. We confirmed that strategies reducing vimentin dynamics, as monitored by FRAP, including cysteine crosslinking and ATP synthesis inhibition, prevent diamide effect. In turn, phosphorylation may promote vimentin disassembly. Indeed, treatment with the phosphatase inhibitor calyculin A to prevent dephosphorylation intensifies electrophile-induced wild-type vimentin filament disruption. However, whereas a phosphorylation-deficient vimentin mutant is only partially protected from disorganization, Cys328Ser vimentin is virtually resistant, even in the presence of calyculin A. Together, these results indicate that modification of Cys328 and vimentin exchange are critical for electrophile-induced network disruption., (Copyright © 2019 Consejo Superior de Investigaciones Científicas (Spain). Published by Elsevier B.V. All rights reserved.)

The nuclear lamins A, B, and C are intermediate filament proteins that form a nuclear scaffold adjacent to the inner nuclear membrane in higher eukaryotes, providing structural support for the nucleus. In the past two decades it has become evident that the final step in the biogenesis of the mature lamin A from its precursor prelamin A by the zinc metalloprotease ZMPSTE24 plays a critical role in human health. Defects in prelamin A processing by ZMPSTE24 result in premature aging disorders including Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid diseases. Additional evidence suggests that defects in prelamin A processing, due to diminished ZMPSTE24 expression or activity, may also drive normal physiological aging. Because of the important connection between prelamin A processing and human aging, there is increasing interest in how ZMPSTE24 specifically recognizes and cleaves its substrate prelamin A, encoded by LMNA. Here, we describe two humanized yeast systems we have recently developed to examine ZMPSTE24 processing of prelamin A. These systems differ from one another slightly. Version 1.0 is optimized to analyze ZMPSTE24 mutations, including disease alleles that may affect the function or stability of the protease. Using this system, we previously showed that some ZMPSTE24 disease alleles that affect stability can be rescued by the proteasome inhibitor bortezomib, which may have therapeutic implications. Version 2.0 is designed to analyze LMNA mutations at or near the ZMPSTE24 processing site to assess whether they permit or impede prelamin A processing. Together these systems offer powerful methodology to study ZMPSTE24 disease alleles and to dissect the specific residues and features of the lamin A tail that are required for recognition and cleavage by the ZMPSTE24 protease., (Copyright © 2019. Published by Elsevier Inc.)

Filaggrin (FLG) and corneodesmosin (CDSN) are two key proteins of the human epidermis. FLG loss-of-function mutations are the strongest genetic risk factors for human atopic dermatitis. Studies of the epidermal distribution of canine FLG and CDSN are limited. Our aim was to better characterize the distribution of FLG and CDSN in canine skin. Using immunohistochemistry on beagle skin, we screened a series of monoclonal antibodies (mAbs) specific for human FLG and CDSN. The cross-reactive mAbs were further used using immunoelectron microscopy and Western blotting. The structure of canine CDSN and FLG was determined using publicly available databases. In the epidermis, four anti-FLG mAbs stained keratohyalin granules in the granular keratinocytes and corneocyte matrix of the lower cornified layer. In urea-extracts of dog epidermis, several bands corresponding to proFLG and FLG monomers were detected. One anti-CDSN mAb stained the cytoplasm of granular keratinocytes and cells of both the inner root sheath and medulla of hair follicles. Dog CDSN was located in lamellar bodies, in the extracellular parts of desmosomes and in corneodesmosomes. A protein of 52 kDa was immunodetected. Genomic DNA analysis revealed that the amino acid sequence and structure of canine and human CDSN were highly similar.

Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.

The interaction of intermediate filaments (IFs) with the cell-cell adhesion complexes desmosomes is crucial for cytoskeletal organization and cell resilience in the epidermis and heart. The intracellular desmosomal protein desmoplakin anchors IFs to the cell adhesion complexes predominantly via its four last carboxy-terminal domains (C-terminus). However, it remains unclear why the C-terminus of desmoplakin interacts with different IF types or if there are different binding affinities for each type of IFs that may influence the stability of cell-specific adhesion complexes. By yeast three-hybrid and fluorescence binding assays, we found that the coiled-coil 1 of the conserved central rod domain of the heterodimeric cytokeratins (Ks) 5 and 14 (K5/K14) was required for their interaction with the C-terminus of desmoplakin, while their unique amino head- and C-tail domains were dispensable. Similar findings were obtained in vitro with K1/K10, and the type III IF proteins desmin and vimentin. Binding assays testing the C-terminus of desmoplakin with assembled K5/K14 and desmin IFs yielded an apparent affinity in the nM range. Our findings reveal that the same conserved domain of IF proteins binds to the C-terminus of desmoplakin, which may help explain the previously reported broad binding IF-specificity to desmoplakin. Our data suggest that desmoplakin high-affinity binding to diverse IF proteins ensures robust linkages of IF cytoskeleton and desmosomes that maintain the structural integrity of cellular adhesion complexes. In summary, our results give new insights into the molecular basis of the IF-desmosome association., Competing Interests: The authors have declared that no competing interests exist.

The barrier function of skin is indispensable for terrestrial animals. This function is mainly carried out by the epidermis, more specifically by its granular and cornified layers. The major structural components associated with this function are the intercellular lipid layer, desmosomes, corneodesmosomes, tight junctions, cornified cell envelope and keratin filaments. In this review, we discuss the current knowledge of their ultrastructure, their molecular basis and their relevance to skin disease., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The type III intermediate filament protein glial fibrillary acidic protein (GFAP) contributes to the homeostasis of astrocytes, where it co-polymerizes with vimentin. Conversely, alterations in GFAP assembly or degradation cause intracellular aggregates linked to astrocyte dysfunction and neurological disease. Moreover, injury and inflammation elicit extensive GFAP organization and expression changes, which underline reactive gliosis. Here we have studied GFAP as a target for modification by electrophilic inflammatory mediators. We show that the GFAP cysteine, C294, is targeted by lipoxidation by cyclopentenone prostaglandins (cyPG) in vitro and in cells. Electrophilic modification of GFAP in cells leads to a striking filament rearrangement, with retraction from the cell periphery and juxtanuclear condensation in thick bundles. Importantly, the C294S mutant is resistant to cyPG addition and filament disruption, thus highlighting the critical role of this residue as a sensor of oxidative damage. However, GFAP C294S shows defective or delayed network formation in GFAP-deficient cells, including SW13/cl.2 cells and GFAP- and vimentin-deficient primary astrocytes. Moreover, GFAP C294S does not effectively integrate with and even disrupts vimentin filaments in the short-term. Interestingly, short-spacer bifunctional cysteine crosslinking produces GFAP-vimentin heterodimers, suggesting that a certain proportion of cysteine residues from both proteins are spatially close. Collectively, these results support that the conserved cysteine residue in type III intermediate filament proteins serves as an electrophilic stress sensor and structural element. Therefore, oxidative modifications of this cysteine could contribute to GFAP disruption or aggregation in pathological situations associated with oxidative or electrophilic stress., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The ubiquitin proteasome system and autophagy are major protein turnover mechanisms in muscle cells, which ensure stemness and muscle fibre maintenance. Muscle cells contain a high proportion of cytoskeletal proteins, which are prone to misfolding and aggregation; pathological processes that are observed in several neuromuscular diseases called proteinopathies. Despite advances in deciphering the mechanisms underlying misfolding and aggregation, little is known about how muscle cells manage cytoskeletal degradation. Here, we describe a process by which muscle cells degrade the misfolded intermediate filament proteins desmin and vimentin by the proteasome. This relies on the MTM1-UBQLN2 complex to recognize and guide these misfolded proteins to the proteasome and occurs prior to aggregate formation. Thus, our data highlight a safeguarding function of the MTM1-UBQLN2 complex that ensures cytoskeletal integrity to avoid proteotoxic aggregate formation.

Background: The objective of the study was to compare the diagnostic yield of home-made ELISA tests based on synthetic chimeric fibrin/filaggrin citrullinated peptides (CFFCPs) with CCP3 and CCP3.1 commercial tests to detect anti-citrullinated protein/peptide antibodies (ACPAs) in rheumatoid arthritis (RA) patients. The prognostic value is also studied in a cohort of patients with early RA. Moreover, we transfer immunological assays from microtiter plates to microarray formats to allow the simultaneous analysis of several peptide sequences and reduce the volume of serum from patients., Methods: The diagnostic study includes: 100 RA patients who fulfilled the 1987 ACR criteria; 100 healthy blood donors; 35 patients with SLE according ACR criteria; 35 patients with PsA fulfilling the Wright and Moll criteria and 30 patients with HCV infection. The prognostic value study includes 50 patients with early RA with follow-up data available. All samples are from outpatients attending the Rheumatology Department of the Hospital Clinic of Barcelona., Results: Similar sensitivity, specificity and predictive values for the diagnosis of RA of CCFCPs compared to CCP3/CCP3.1 were obtained. Although a high concordance is observed between anti-CFFCPs and anti-CCP3/CCP3.1 in the early patients that rendered Larsen radiographic progression, CFFCPs could be a better marker of radiographic outcome. Strong correlations between the microarray and ELISA results were found for individual CFFCPs peptides., Conclusions: The development of multiplexing techniques combining a different spectrum of markers in a single analysis, including CFFCP peptides, could allow a more detailed analysis of the autoantibodies reactivity found in the sera of patients suffering of this heterogeneous disease.

The growth of hairs occurs during the anagen phase of the follicle cycle. Hair growth begins with basement membrane-bound stem cells (mother cells) around the dermal papilla neck which continuously bud off daughter cells which further divide as a transient amplifying population. Division ceases as cell line differentiation begins, which entails changes in cell junctions, cell shape and position, and cell-line specific cytoplasmic expression of keratin and trichohyalin. As the differentiating cells migrate up the bulb, nuclear function ceases in cortex, cuticle and inner root sheath (IRS) layers. Past the top of the bulb, cell shape/position changes cease, and there is a period of keratin and keratin-associated protein (KAP) synthesis in fibre cell lines, with increases, in particular of KAP species. A gradual keratinization process begins in the cortex at this point and then non-keratin cell components are increasingly broken down. Terminal cornification, or hardening, is associated with water loss and precipitation of keratin. In the upper follicle, the hair, now in its mature form, detaches from the IRS, which is then extracted of material and becomes fragmented to release the fibre. Finally, the sebaceous and sudoriferous (if present) glands coat the fibre in lipid-rich material and the fibre emerges from the skin. This chapter follows the origin of the hair growth in the lower bulb and traces the development of the various cell lines.

Background: Deimination (also known as citrullination), the conversion of arginine in a protein to citrulline, is catalyzed by a family of enzymes called peptidylarginine deiminases (PADs). Three PADs are expressed in the epidermis, one of their targets being filaggrin. Filaggrin plays a central role in atopic dermatitis and is a key protein for the epidermal barrier. It aggregates keratins and is cross-linked to cornified envelopes. Following its deimination, it is totally degraded to release free amino acids, contributing to the natural moisturizing factor (NMF). The mechanisms controlling this multistep catabolism in human are unknown., Objective: To test whether external humidity plays a role, and investigate the molecular mechanisms involved., Methods: Specimens of reconstructed human epidermis (RHEs) produced in humid or dry conditions (>95% or 30-50% relative humidity) were compared., Results: RHEs produced in the dry condition presented structural changes, including a thicker stratum corneum and a larger amount of keratohyalin granules. The transepidermal water loss and the stratum corneum pH were decreased whereas the quantity of NMF was greater. This highly suggested that filaggrin proteolysis was up-regulated. The expression/activity of the proteases involved in filaggrin breakdown did not increase while PAD1 expression and the deimination rate of proteins, including filaggrin, were drastically enhanced. Partial inhibition of PADs with Cl-amidine reversed the effect of dryness on filaggrin breakdown., Conclusion: These results demonstrate the importance of external humidity in the control of human filaggrin metabolism, and suggest that deimination plays a major role in this regulation., (Copyright © 2017 Japanese Society for Investigative Dermatology. All rights reserved.)

The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin. Besides providing structural stability to the nucleus, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication. However, the structural organization of the nuclear lamina is poorly understood. Here we use cryo-electron tomography to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fibre appearance and shows that A- and B-type lamins assemble into tetrameric filaments of 3.5 nm thickness. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshworks at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.

Background: The evolutionary dynamics of repeat sequences is quite complex, with some duplicates never having differentiated from each other. Two models can explain the complex evolutionary process for repeated genes-concerted and birth-and-death, of which the latter is driven by duplications maintained by selection. Copy number variations caused by random duplications and losses in repeat regions may modulate molecular pathways and therefore affect phenotypic characteristics in a population, resulting in individuals that are able to adapt to new environments. In this study, we investigated the filaggrin gene (FLG), which codes for filaggrin-an important component of the outer layers of mammalian skin-and contains tandem repeats that exhibit copy number variation between and within species. To examine which model best fits the evolutionary pathway for the complete tandem repeats within a single exon of FLG, we determined the repeat sequences in crab-eating macaque (Macaca fascicularis), orangutan (Pongo abelii), gorilla (Gorilla gorilla), and chimpanzee (Pan troglodytes) and compared these with the sequence in human (Homo sapiens)., Results: In this study we compared concerted and birth-and-death evolution models, commonly used for gene copies. We found that there is high nucleotide diversity between filaggrin repeat regions, which fits the birth-and-death model. Phylogenetic analyses also suggested that independent duplication events created the repeat sequences in crab-eating macaques and orangutans, while different duplication and loss events created the repeats in gorillas, chimpanzees, and humans. Comparison of the repeat sequences detected purifying selection within species and lineage-specific duplications across species. We also found variation in the length of the repeated region within species such as chimpanzee and crab-eating macaque., Conclusions: We conclude that the copy number variation in the repeat sequences of FLG between primates may be a consequence of species-specific divergence and expansion.

Intermediate filaments (IFs), together with microtubules and actin microfilaments, are the three main cytoskeletal components in metazoan cells. IFs are formed by a distinct protein family, which is made up of 70 members in humans. Most IF proteins are tissue- or organelle-specific, which includes lamins, the IF proteins of the nucleus. The building block of IFs is an elongated dimer, which consists of a central α-helical 'rod' domain flanked by flexible N- and C-terminal domains. The conserved rod domain is the 'signature feature' of the IF family. Bioinformatics analysis reveals that the rod domain of all IF proteins contains three α-helical segments of largely conserved length, interconnected by linkers. Moreover, there is a conserved pattern of hydrophobic repeats within each segment, which includes heptads and hendecads. This defines the presence of both left-handed and almost parallel coiled-coil regions along the rod length. Using X-ray crystallography on multiple overlapping fragments of IF proteins, the atomic structure of the nearly complete rod domain has been determined. Here, we discuss some specific challenges of this procedure, such as crystallization and diffraction data phasing by molecular replacement. Further insights into the structure of the coiled coil and the terminal domains have been obtained using electron paramagnetic resonance measurements on the full-length protein, with spin labels attached at specific positions. This atomic resolution information, as well as further interesting findings, such as the variation of the coiled-coil stability along the rod length, provide clues towards interpreting the data on IF assembly, collected by a range of methods. However, a full description of this process at the molecular level is not yet at hand.

Rheumatoid arthritis (RA) is an autoimmune disease of complex etiology. A characteristic feature of a subset of RA is the presence of anti-citrullinated protein antibodies (ACPA), which correlate with a progressive disease course. In this study, we employed streptavidin capture enzyme-linked immunosorbent assay to analyze ACPA reactivity. Using the pro-filaggrin peptide HQCHQEST-Cit-GRSRGRCGRSGS, as template, we analyzed the reactivity of RA sera and healthy donor sera to various peptides in order to determine the physical characteristics of the citrullinated pro-filaggrin epitope and to examine whether biotin labelling influence antibody recognition. The full-length cyclic pro-filaggrin peptide and a linear form with a N-terminal biotin, was recognized to the same level, whereas, a notable difference in ACPA reactivity to the linear peptides with a C-terminal biotin was found, probably due to steric hindrance. Screening of linear and cyclic truncated peptides, revealed that small cyclic peptides containing 10-12 amino acids are favored over the linear. Moreover, the charged amino acids C-terminal to citrulline were found to be essential for antibody reactivity, most important was the charged amino acid in position 4 C-terminal to citrulline. Collectively, peptide structure, length, the presence of charged amino acids and biotin labelling markedly influence antibody reactivity. In relation to the clinical diagnostics of ACPA, these findings may reflect the differences in diagnostic assays used for detection of ACPA, which relates to differences in sensitivity and specificity dependent on the assay applied., Competing Interests: The authors have declared that no competing interests exist.

α-Internexin is a member of the neuronal intermediate filament (nIF) protein family, which also includes peripherin and neurofilament (NF) triplet proteins. Previous studies found that expression of α-internexin precedes that of the NF triplet proteins in mammals and suggested that α-internexin plays a key role in the neuronal cytoskeleton network during development. In this study, we aimed to analyze the expression patterns and function of internexin neuronal intermediate filament protein-alpha a (inaa), the encoding gene of which is a homolog of the mammalian α-internexin, during retinal development in zebrafish. Via in vitro and in vivo studies, we demonstrated that zebrafish inaa is an α-internexin homolog that shares characteristics with nIFs. An immunohistochemical analysis of zebrafish revealed that inaa was distributed dynamically in the developing retina. It was widely localized in retinal neuroepithelial cells at 1 day postfertilization (dpf), and was mainly found in the ganglion cell layer (GCL) and inner part of the inner nuclear layer (INL) from 3-9 dpf; after 14 dpf, it was restricted to the outer nuclear layer (ONL). Moreover, we demonstrated for the first time that inaa acted distinctively from the cytoskeletal scaffold of zebrafish cone photoreceptors during development. In conclusion, we demonstrated the morphological features of a novel nIF, inaa, and illustrated its developmental expression pattern in the zebrafish retina. J. Comp. Neurol. 524:3810-3826, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: GFAP is the major intermediate filament protein in mature astrocytes. Its increased expression and aggregation was firstly associated to Alexander's disease, and successively in different neurological diseases including scrapie, Alzheimer's and Creutzfeld-Jacob diseases. Recently, ceftriaxone a multi-potent β-lactam antibiotic able to overcome the blood-brain barrier, successfully eliminated the cellular toxic effects of misfolded mutated GFAP, similarly to phenytoin sodium, in a cellular model of Alexander's disease and inhibited α-synuclein aggregation protecting PC12 cells from the exposure to 6-hydroxydopamine., Methods: In this study, synchrotron radiation circular dichroism spectroscopy has been used to obtain structural information about the GFAP-ceftriaxone (phenytoin) interactions, while computational methods allowed the identification of the relevant putative binding site of either ceftriaxone or phenytoin on the dimer structure of GFAP, permitting to rationalize the spectroscopic experimental results., Results: We found that GFAP exhibited enhanced stability upon the addition of two equivalents of each ligands with ceftriaxone imparting a more spontaneous interactions and a more ordered complex system than phenytoin., Conclusions: SRCD data and MD models indicate a stronger protective effect of ceftriaxone in neurological disorders characterized by an increased production and polymerization of GFAP., General Significance: This result, in addition to our previous works in which we documented that ceftriaxone interacts with α-synuclein inhibiting its pathological aggregation and that a cyclical treatment with this molecule in a patient with adult-onset Alexander's disease halted, and partly reversed, the progression of neurodegeneration, suggests the possibility of a chaperone-like effect of ceftriaxone on protein involved in specific neurodegenerative diseases., (Copyright © 2016 Diamond Light Source Ltd. Published by Elsevier B.V. All rights reserved.)

Neurofilaments are intermediate filament proteins specific for neurons and characterized by formation of biochemically stable, obligate heteropolymers in vivo While purified or reassembled neurofilaments have been subjected to morphological analyses by electron microscopy and atomic force microscopy, there has been a need for direct imaging of cytoplasmic genuine intermediate filaments with minimal risk of artefactualization. In this study, we applied the modified 'cells on glass sandwich' method to exteriorize intracellular neurofilaments, reducing the risk of causing artefacts through sample preparation. SW13vim(-) cells were double transduced with neurofilament medium polypeptide (NF-M) and alpha-internexin (α-inx). Cultured cells were covered with a cationized coverslip after prestabilization with tannic acid to form a sandwich and then split into two. After confirming that neurofilaments could be deposited on ventral plasma membranes exposed via unroofing, we performed atomic force microscopy imaging semi-in situ in aqueous solution. The observed thin filaments, considered to retain native structures of the neurofilaments, exhibited an approximate periodicity of 50-60 nm along their length. Their structural property appeared to reflect the morphology formed by their constituents, i.e. NF-M and α-inx. The success of semi-in situ atomic force microscopy of exposed bona fide assembled neurofilaments through separating the sandwich suggests that it can be an effective and alternative method for investigating cytoplasmic intermediate filaments under physiological conditions by atomic force microscopy., (© The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

Background: Peptidylarginine deiminases (PADs) catalyze deimination (or citrullination), a calcium-dependent post-translational modification involved in several physiological processes and human diseases, such as rheumatoid arthritis and cancer. Deimination of filaggrin (FLG) by PAD1 and PAD3 during the last steps of keratinocyte differentiation is a crucial event for the epidermis function and homeostasis. This allows the complete degradation of FLG, leading to the production of free amino acids and their derivatives that are essential for epidermal photoprotection and moisturizing of the stratum corneum., Objective: To increase the flux of this catabolic pathway, we searched for activators of PADs., Methods: A large chemical library was screened first in silico and then by using an automated assay based on an indirect colorimetric measurement of recombinant human PAD activity. Potential activators were then confirmed using a recombinant human FLG as a substrate, and secondly after topical application at the surface of three-dimensional reconstructed human epidermis., Results: The data obtained after the library screening pointed to xanthine derivatives as potential PAD activators. Among seven xanthine derivatives tested at 50-300μM, caffeine, theobromine and acefylline proved to be the most potent enhancers of in vitro deimination of FLG by PAD1 and PAD3. After topical application of a gel formulation containing 3% acefylline at the surface of reconstructed epidermis, immunoblotting analysis showed an increase in the total amount of deiminated proteins, and confocal microscopy showed an enhanced deimination in the stratum corneum. This demonstrated the activation of PADs in living cells., Conclusion: As a PAD activator, acefylline will be useful to study the role of deimination and could be proposed to increase or correct the hydration of the cornified layers of the epidermis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Desmoplakin (DP) is a cytoskeletal linker protein that connects the desmosomal cadherin/plakoglobin/plakophilin complex to intermediate filaments (IFs). The C-terminal region of DP (DPCT) mediates IF binding, and contains three plakin repeat domains (PRDs), termed PRD-A, PRD-B and PRD-C. Previous crystal structures of PRDs B and C revealed that each is formed by 4.5 copies of a plakin repeat (PR) and has a conserved positively charged groove on its surface. Although PRDs A and B are linked by just four amino acids, B and C are separated by a 154 residue flexible linker, which has hindered crystallographic analysis of the full DPCT. Here we present the crystal structure of a DPCT fragment spanning PRDs A and B, and elucidate the overall architecture of DPCT by small angle X-ray scattering (SAXS) analysis. The structure of PRD-A is similar to that of PRD-B, and the two domains are arranged in a quasi-linear arrangement, and separated by a 4 amino acid linker. Analysis of the B-C linker region using secondary structure prediction and the crystal structure of a homologous linker from the cytolinker periplakin suggests that the N-terminal ~100 amino acids of the linker form two PR-like motifs. SAXS analysis of DPCT indicates an elongated but non-linear shape with Rg = 51.5 Å and Dmax = 178 Å. These data provide the first structural insights into an IF binding protein containing multiple PRDs and provide a foundation for studying the molecular basis of DP-IF interactions.

Hagfish slime is an ultra dilute, elastic and cohesive hydrogel that deploys within milliseconds in cold seawater from a glandularly secreted exudate. The slime is made of long keratin-like fibers and mucin-like glycoproteins that span a network which entraps water and acts as a defense mechanism against predators. Unlike other hydrogels, the slime only confines water physically and is very susceptible to mechanical stress, which makes it unsuitable for many processing operations and potential applications. Despite its huge potential, little work has been done to improve and functionalize the properties of this hydrogel. To address this shortcoming, hagfish exudate was mixed with a soy protein isolate suspension (4% w/v) and with a soy emulsion (commercial soy milk) to form a more stable structure and combine the functionalities of a suspension and emulsion with those of the hydrogel. Hagfish exudate interacted strongly with the soy systems, showing a markedly increased viscoelasticity and water retention. Hagfish mucin was found to induce a depletion and bridging mechanism, which caused the emulsion and suspension to flocculate, making "soy slime", a cohesive and cold-set emulsion- and particle gel. The flocculation network increases viscoelasticity and substantially contributes to liquid retention by entrapping liquid in the additional confinements between aggregated particles and protein fibers. Because the mucin-induced flocculation resembles the salt- or acid-induced flocculation in tofu curd production, the soy slime was cooked for comparison. The cooked soy slime was similar to conventional cooked tofu, but possessed a long-range cohesiveness from the fibers. The fibrous, cold-set, and curd-like structure of the soy slime represents a novel way for a cold coagulation and fiber incorporation into a suspension or emulsion. This mechanism could be used to efficiently gel functionalized emulsions or produce novel tofu-like structured food products.

Experimental and theoretical research aimed at determining the structure and function of the family of intermediate filament proteins has made significant advances over the past 20 years. Much of this has either contributed to or relied on the amino acid sequence databases that are now available online, and the data mining approaches that have been developed to analyze these sequences. As the quality of sequence data is generally high, it follows that it is the design of the computational and graphical methodologies that are of especial importance to researchers who aspire to gain a greater understanding of those sequence features that specify both function and structural hierarchy. However, these techniques are necessarily subject to limitations and it is important that these be recognized. In addition, no single method is likely to be successful in solving a particular problem, and a coordinated approach using a suite of methods is generally required. A final step in the process involves the interpretation of the results obtained and the construction of a working model or hypothesis that suggests further experimentation. While such methods allow meaningful progress to be made it is still important that the data are interpreted correctly and conservatively. New data mining methods are continually being developed, and it can be expected that even greater understanding of the relationship between structure and function will be gleaned from sequence data in the coming years., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purified intermediate filament (IF) proteins can be reassembled in vitro to produce polymers closely resembling those found in cells, and these filaments form viscoelastic gels. The cross-links holding IFs together in the network include specific bonds between polypeptides extending from the filament surface and ionic interactions mediated by divalent cations. IF networks exhibit striking nonlinear elasticity with stiffness, as quantified by shear modulus, increasing an order of magnitude as the networks are deformed to large strains resembling those that soft tissues undergo in vivo. Individual IFs can be stretched to more than two or three times their resting length without breaking. At least 10 different rheometric methods have been used to quantify the viscoelasticity of IF networks over a wide range of timescales and strain magnitudes. The mechanical roles of different classes of cytoplasmic IFs on mesenchymal and epithelial cells in culture have also been studied by an even wider range of microrheological methods. These studies have documented the effects on cell mechanics when IFs are genetically or pharmacologically disrupted or when normal or mutant IF proteins are exogenously expressed in cells. Consistent with in vitro rheology, the mechanical role of IFs is more apparent as cells are subjected to larger and more frequent deformations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Intermediate 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., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, we conducted a global fluorescence-based screen to reveal endomembrane effector genes. Screening implicated Phox (PX) domain-containing protein Mdm1 in endomembrane dynamics. Surprisingly, we demonstrate that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)-vacuole/lysosome membrane contact sites (MCSs). We show that Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER-vacuole hypertethering, underscoring its role as an interorganelle tether. We also show that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER-vacuole MCSs independently of established tether Nvj1. Finally, we find that Mdm1 truncations analogous to neurological disease-associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. Our work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism., (© 2015 Henne et al.)

Neuronal cytoplasmic intermediate filaments are principal structural and mechanical elements of the axon. Their expression during embryonic development follows a differential pattern, while their unregulated expression is correlated to neurodegenerative diseases. The largest neurofilament proteins of medium (NF-M) and high molecular weight (NF-H) were shown to modulate the axonal architecture and inter-filament spacing. However, the individual roles of the remaining α-internexin (α-Inx) and neurofilament of low molecular weight (NF-L) proteins in composite filaments remained elusive. In contrast to previous predictions, we show that when co-assembled with NF-M, the shortest and the least charged α-Inx protein increases inter-filament spacing. These findings suggest a novel structural explanation for the expression pattern of neurofilament proteins during embryonic development. We explain our results by an analysis of ionic cross-links between the disordered polyampholytic C-terminal tails and suggest that a collapsed conformation of the α-Inx tail domain interferes with tail cross-linking near the filament backbone.

The nuclear lamina is a protein meshwork associated with the inner side of the nuclear envelope contributing structural, signalling and regulatory functions. Here, I report on the evolution of an important component of the lamina, the lamin intermediate filament proteins, across the eukaryotic tree of life. The lamins show a variety of protein domain and sequence motif architectures beyond the classical α-helical rod, nuclear localisation signal, immunoglobulin domain and CaaX motif organisation, suggesting extension and adaptation of functions in many species. I identified lamin genes not only in metazoa and Amoebozoa as previously described, but also in other opisthokonts including Ichthyosporea and choanoflagellates, in oomycetes, a sub-family of Stramenopiles, and in Rhizaria, implying that they must have been present very early in eukaryotic evolution if not even the last common ancestor of all extant eukaryotes. These data considerably extend the current perception of lamin evolution and have important implications with regard to the evolution of the nuclear envelope.

The intermediate filament protein IFA-2 is essential for the structural integrity of the Caenorhabditis elegans epidermis. It is one of the major components of the fibrous organelle, an epidermal structure comprised of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments that serve to transmit force from the muscle to the cuticle. Mutations of IFA-2 result in epidermal fragility and separation of the apical and basal epidermal surfaces during postembryonic growth. An IFA-2 lacking the head domain fully rescues the IFA-2 null mutant, whereas an IFA-2 lacking the tail domain cannot. Conversely, an isolated IFA-2 head was able to localize to fibrous organelles whereas the tail was not. Taken together these results suggest that the head domain contains redundant signals for IF localization, whereas non-redundant essential functions map to the IFA-2, tail, although the tail is unlikely to be directly involved in fibrous organelle localization.

Studies on the assembly of neuronal intermediate filaments (IFs) date back to the early work of Alzheimer. Developing neurons express a series of IF proteins, sequentially, at distinct stages of mammalian cell differentiation. This correlates with altered morphologies during the neuronal development, including axon outgrowth, guidance and conductivity. Importantly, neuronal IFs that fail to properly assemble into a filamentous network are a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's, and Parkinson's disease. Traditional structural methodologies fail to fully describe neuronal IF assembly, interactions and resulting function due to IFs structural plasticity, particularly in their C-terminal domains. We review here current progress in the field of neuronal-specific IFs, a dominant component affecting the cytoskeletal structure and function of neurons., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Intermediate filament (IF) proteins, together with tubulins and actins, constitute the majority of cytoskeletal proteins in metazoans. Proteins of the IF family fulfil increasingly diverse functions but share common structural features. Phylogenetic analysis within the metazoan lineage traces back their origin to a common lamin-like ancestor. Major steps in lamin evolution occurred at the base of the vertebrate radiation, while cytoplasmic IF protein subfamilies evolved independently in the major metazoan lineages., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cell migration is a multistep process which relies on the coordination of cytoskeletal structures in space and time. While the roles of actin and microtubules have been investigated in great details, the lack of inhibitors and visualizing tools and the large number of proteins forming intermediate filaments (IFs) have delayed the characterization of IF functions during migration. However, a large body of evidence has progressively pointed to changes in IF composition as an important parameter in the regulation of cell migratory properties both during development and tumor invasion. More recent in-depth analyses show that IFs are dynamically reorganized to participate, together with microfilaments and microtubules, to the key steps leading to cell migration., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The tight junction (TJ) barrier is located in the granular layer of the epidermis. Filaggrin deficiency predisposes patients to atopic dermatitis (AD) by impairing stratum corneum (SC) barrier function. Altered TJ barrier function has been observed in the skin of patients with AD; however, it remains unclear whether TJ function is influenced by filaggrin deficiency directly or secondarily via skin inflammation., Objective: To investigate the in vivo effects of filaggrin deficiency and skin inflammation on epidermal TJ function., Methods: Morphological changes in the TJ were investigated in filaggrin knockout mice and mice with hapten-induced dermatitis using en face visualization of epidermal sheets, and functional changes in the TJ were assessed with an in vivo permeation assay using tracers of various sizes., Results: In filaggrin knockout mice, there was no apparent change in the honeycomb morphology of the TJ, TJ component mRNA expression, or TJ barrier function in neonates and adults, indicating that filaggrin-deficiency had no direct effects on the TJ. By contrast, in mice with hapten-induced dermatitis, the mRNA expression of TJ components was decreased markedly and the TJ barrier function was size-dependently impaired: the TJ leaked small tracers (<5 kDa), but not large tracers (>30 kDa)., Conclusion: Filaggrin deficiency did not affect the epidermal TJ barrier directly, but once dermatitis occurred, the skin inflammation induced TJ dysfunction. Since TJ dysfunction induces the SC barrier impairment, skin inflammation will enhance skin permeability to external antigens and result in a vicious cycle of barrier dysfunction and skin inflammation., (Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)