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Islet β-cell dysfunction is an underlying factor for type I diabetes (T1D) development. Insulin sensing and secretion are tightly regulated in β-cells at multiple subcellular levels. The epithelial intermediate filament (IF) protein keratin (K) 8 is the main β-cell keratin, constituting the filament network with K18. To identify the cell-autonomous functions of K8 in β-cells, mice with targeted deletion of β-cell K8 (K8flox/flox; Ins-Cre) were analyzed for islet morphology, ultrastructure, and integrity, as well as blood glucose regulation and streptozotocin (STZ)-induced diabetes development. Glucose transporter 2 (GLUT2) localization was studied in β-cells in vivo and in MIN6 cells with intact or disrupted K8/K18 filaments. Loss of β-cell K8 leads to a major reduction in K18. Islets without β-cell K8 are more fragile, and these β-cells display disjointed plasma membrane organization with less membranous E-cadherin and smaller mitochondria with diffuse cristae. Lack of β-cell K8 also leads to a reduced glucose-stimulated insulin secretion (GSIS) response in vivo, despite undisturbed systemic blood glucose regulation. K8flox/flox, Ins-Cre mice have a decreased sensitivity to STZ compared with K8 wild-type mice, which is in line with decreased membranous GLUT2 expression observed in vivo, as GLUT2 is required for STZ uptake in β-cells. In vitro, MIN6 cell plasma membrane GLUT2 is rescued in cells overexpressing K8/K18 filaments but mistargeted in cells with disrupted K8/K18 filaments. β-Cell K8 is required for islet and β-cell structural integrity, normal mitochondrial morphology, and GLUT2 plasma membrane targeting, and has implications on STZ sensitivity as well as systemic insulin responses. NEW & NOTEWORTHY: Keratin 8 is the main cytoskeletal protein in the cytoplasmic intermediate filament network in β-cells. Here for the first time, we assessed the β-cell autonomous mechanical and nonmechanical roles of keratin 8 in β-cell function. We demonstrated the importance of keratin 8 in islet and β-cell structural integrity, maintaining mitochondrial morphology and GLUT2 plasma membrane targeting. [ABSTRACT FROM AUTHOR]

Desmin, the most abundant intermediate filament in cardiomyocytes, plays a key role in maintaining cardiomyocyte structure by interconnecting intracellular organelles, and facilitating cardiomyocyte interactions with the extracellular matrix and neighboring cardiomyocytes. As a consequence, mutations in the desmin gene (DES) can lead to desminopathies, a group of diseases characterized by variable and often severe cardiomyopathies along with skeletal muscle disorders. The basic desmin intermediate filament structure is composed of four segments separated by linkers that further assemble into dimers, tetramers and eventually unit‐length filaments that compact radially to give the final form of the filament. Each step in this process is critical for proper filament formation and allow specific interactions within the cell. Mutations within the desmin gene can disrupt filament formation, as seen by aggregate formation, and thus have severe cardiac and skeletal outcomes, depending on the locus of the mutation. The focus of this review is to outline the cardiac molecular consequences of mutations located in the C‐terminal part of segment 2B. This region is crucial for ensuring proper desmin filament formation and is a known hotspot for mutations that significantly impact cardiac function. [ABSTRACT FROM AUTHOR]

Filensin and phakinin are lens fiber cell‐specific proteins that constitute the beaded filaments (BFs) that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N‐ and C‐terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize the effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30–416) and Fil(30–369), respectively, were examined. Fil(30–416) lacks the N‐terminal 29 amino acids and the C‐terminal 248 amino acids. Fil(30–369) lacks the N‐terminal 29 residues and the C‐terminal 295 residues. In cell‐free assembly characterized by electron microscopy, filensin and Fil(30–416) co‐polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30–369) formed only aggregates. In cultured SW‐13 and MCF‐7 cells expressing fluorescent fusion proteins, filensin and Fil(30–416) co‐polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30–369) gave aggregates. Therefore, while truncation of the N‐terminal 29 amino acids did not affect filament formation, truncation of the C‐terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370–416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with BF formation and thereby contribute to opacification.

ARL15, a small GTPase protein, was linked to metabolic traits in association studies. We aimed to test the Arl15 gene as a functional candidate for metabolic traits in the mouse. CRISPR/Cas9 germline knockout (KO) of Arl15 showed that homozygotes were postnatal lethal and exhibited a complete cleft palate (CP). Also, decreased cell migration was observed from Arl15 KO mouse embryonic fibroblasts (MEFs). Metabolic phenotyping of heterozygotes showed that females had reduced fat mass on a chow diet from 14 weeks of age. Mild body composition phenotypes were also observed in heterozygous mice on a high‐fat diet (HFD)/low‐fat diet (LFD). Females on a HFD showed reduced body weight, gonadal fat depot weight and brown adipose tissue (BAT) weight. In contrast, in the LFD group, females showed increased bone mineral density (BMD), while males showed a trend toward reduced BMD. Clinical biochemistry analysis of plasma on HFD showed transient lower adiponectin at 20 weeks of age in females. Urinary and plasma Mg2+ concentrations were not significantly different. Our phenotyping data showed that Arl15 is essential for craniofacial development. Adult metabolic phenotyping revealed potential roles in brown adipose tissue and bone development. [ABSTRACT FROM AUTHOR]

We identified 23 and 20 cytoplasmic IF (cIF) genes in the two Branchiostoma belcheri and Branchiostoma lanceolatum cephalochordates, respectively. Combining these results with earlier data on the related Branchiostoma floridae, the following conclusions can be drawn. First, the Branchiostoma N4 protein with a long lamin-like coil 1B segment is the only protostomic-type cIF found so far in any analysed chordate or vertebrate organism. Second, Branchiostoma is the only organism known so far containing both the long protostomic- and the short chordate-prototypes of cIFs. This finding provides so far missing molecular evidence for the phylogenetic transition between the protostomic- and the chordate-type IF sequences at the base of the cephalochordates and vertebrates. Third, this finding also brings some support for another hypothesis, that the long protostomic-type cIF is subjected to evolutionary constraints in order to preclude inappropriate interactions with lamin and that the latter complexes might be prevented by a several heptad-long rod deletion, which released the selective constraints on it and promoted, at least in part, its expansion in nematodes, cephalochordates, and in vertebrates. Finally, here-presented data confirmed our previous results that cephalochordates do not have any vertebrate type III or type IV IF homolog. [ABSTRACT FROM AUTHOR]

Filensin and phakinin are lens fiber cell‐specific proteins that constitute the beaded filaments (BFs) that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N‐ and C‐terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize the effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30–416) and Fil(30–369), respectively, were examined. Fil(30–416) lacks the N‐terminal 29 amino acids and the C‐terminal 248 amino acids. Fil(30–369) lacks the N‐terminal 29 residues and the C‐terminal 295 residues. In cell‐free assembly characterized by electron microscopy, filensin and Fil(30–416) co‐polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30–369) formed only aggregates. In cultured SW‐13 and MCF‐7 cells expressing fluorescent fusion proteins, filensin and Fil(30–416) co‐polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30–369) gave aggregates. Therefore, while truncation of the N‐terminal 29 amino acids did not affect filament formation, truncation of the C‐terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370–416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with BF formation and thereby contribute to opacification. [ABSTRACT FROM AUTHOR]

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process. [ABSTRACT FROM AUTHOR]

We have investigated how connexin 46 (Cx46) regulates lens stiffness by studying different Cx46 knockout (Cx46KO) mice. A modified muscle lever system was used to determine the lens stiffness of wild-type (WT) and Cx46KO mice at the C57BL/6J (B6) and the 129SvJae (129) strain backgrounds according to total lens displacement at the point of maximum force when fresh lenses were compressed with a maximum of 2 mN of force. In comparison to B6-WT controls, young and old B6-Cx46KO lenses showed 23% and 28% reductions in lens displacement, respectively. Comparing to 129-WT controls, old 129-Cx46KO lenses showed 50% reduction in the lens displacement while young 129-Cx46KO lenses displayed similar displacement. Old B6-Cx46KO and old 129-Cx46KO lenses showed almost identical lens displacement, 128 μm versus 127 μm. Morphological data revealed unique changes of peripheral fiber cell shapes in young B6-WT lenses but not in young B6-Cx46KO, 129-WT and 129-Cx46KO lenses. This work reveals Cx46 deletion increases the lens stiffness in both young and old mice at B6 strain background but only in old mice at 129 strain background which contains intermediate filament CP49 gene deletion. Cx46 impairment increases old mouse lens stiffness and may contribute to the development of presbyopia.

During eukaryotic cell division, organelles are distributed between daughter cells through a dynamic process to ensure that cells can differentiate and perform their functions correctly. Uncovering the mode of lipid droplet (LD) distribution may help reveal the mechanism of membrane remodeling during cell division and lipid droplet function. Our results showed that LDs were equally distributed in both daughter cells during cytokinesis. Further experiments demonstrated that the key factor regulating the movement of LDs is the microtubule (MT)-resident protein KIF5B. Because the KIF5B structure lacks a hydrophilic region, we believe that there are proteins that mediate the interaction between LDs and KIF5B. Mass spectrometric detection of KIF5B-interacting proteins on the surface of LDs demonstrated that LDs were first wrapped by intermediate filaments forming a meshwork and then contacted with MTs to mediate lipid droplet movement during cytokinesis. Disruption of the homogeneous distribution of LDs may hinder cell proliferation and even lead to apoptosis. [ABSTRACT FROM AUTHOR]

Research on pathogenic mechanisms underlying giant axonal neuropathy (GAN), a disease caused by a deficiency of gigaxonin, has been hindered by the lack of appropriate animal models exhibiting substantial symptoms and large neurofilament (NF) swellings, a hallmark of the human disease. It is well established that intermediate filament (IF) proteins are substrates for gigaxonin-mediated degradation. However, it has remained unknown to what extent NF accumulations contribute to GAN pathogenesis. Here, we report the generation of a new mouse model of GAN that is based on crossing transgenic mice overexpressing peripherin (Prph) with mice knockout for Gan. The Gan2/2;TgPer mice developed early onset sensory-motor deficits along with IF accumulations made up of NF proteins and of Prph, causing swelling of spinal neurons at a young age. Abundant inclusion bodies composed of disorganized IFs were also detected in the brain of Gan2/2;TgPer mice. At 12months of age, the Gan2/2;TgPer mice exhibited cognitive deficits as well as severe sensory and motor defects. The disease was associated with neuroinflammation and substantial loss of cortical neurons and spinal neurons. Giant axons (-160 lm2) enlarged by disorganized IFs, a hallmark of GAN disease, were also detected in dorsal and ventral roots of the Gan2/2;TgPer mice. These results, obtained with both sexes, support the view that the disorganization of IFs can drive some neurodegenerative changes caused by gigaxonin deficiency. This new mouse model should be useful to investigate the pathogenic changes associated with GAN disease and for drug testing. [ABSTRACT FROM AUTHOR]

Intermediate filaments (IFs) are major components of the metazoan cytoskeleton. A long-standing debate concerns the question whether IF network organization only reflects or also determines cell and tissue function. Using Caenorhabditis elegans, we have recently described mutants of the mitogen-activated protein kinase (MAPK) SMA-5 which perturb the organization of the intestinal IF cytoskeleton resulting in luminal widening and cytoplasmic invaginations. Besides these structural phenotypes, systemic dysfunctions were also observed. We now identify the IF polypeptide IFB-2 as a highly efficient suppressor of both the structural and functional deficiencies of mutant sma-5 animals by removing the aberrant IF network. Mechanistically, perturbed IF network morphogenesis is linked to hyperphosphorylation of multiple sites throughout the entire IFB-2 molecule. The rescuing capability is IF isotype-specific and not restricted to sma-5 mutants but extends to mutants that disrupt the function of the cytoskeletal linker IFO-1 and the IF-associated protein BBLN-1. The findings provide strong evidence for adverse consequences of the deranged IF networks with implications for diseases that are characterized by altered IF network organization.

The cytoskeleton comprises three polymerizing structures that have been studied for a long time, actin microfilaments, microtubules and intermediate filaments, plus more recently investigated dynamic assemblies like septins or the endocytic-sorting complex required for transport (ESCRT) complex. These filament-forming proteins control several cell functions through crosstalks with each other and with membranes. In this review, we report recent works that address how septins bind to membranes, and influence their shaping, organization, properties and functions, either by binding to them directly or indirectly through other cytoskeleton elements.

A large group of keratin genes (n=54 in the human genome) code for intermediate filament (IF)-forming proteins and show differential regulation in epithelial cells and tissues. Keratin expression can be highly informative about the type of epithelial tissue, differentiation status of constituent cells and biological context (e.g. normal versus diseased settings). The foundational principles underlying the use of keratin expression to gain insight about epithelial cells and tissues primarily originated in pioneering studies conducted in the 1980s. The recent emergence of single cell transcriptomics provides an opportunity to revisit these principles and gain new insight into epithelial biology. Re-analysis of single-cell RNAseq data collected from human and mouse skin has confirmed long-held views regarding the quantitative importance and pairwise regulation of specific keratin genes in keratinocytes of surface epithelia. Furthermore, such analyses confirm and extend the notion that changes in keratin gene expression occur gradually as progenitor keratinocytes commit to and undergo differentiation, and challenge the prevailing assumption that specific keratin combinations reflect a mitotic versus a post-mitotic differentiating state. Our findings provide a blueprint for similar analyses in other tissues, and warrant a more nuanced approach in the use of keratin genes as biomarkers in epithelia. [ABSTRACT FROM AUTHOR]

Abstract Intermediate filament (IntFil) genes arose during early metazoan evolution, to provide mechanical support for plasma membranes contacting/interacting with other cells and the extracellular matrix. Keratin genes comprise the largest subset of IntFil genes. Whereas the first keratin gene appeared in sponge, and three genes in arthropods, more rapid increases in keratin genes occurred in lungfish and amphibian genomes, concomitant with land animal-sea animal divergence (~ 440 to 410 million years ago). Human, mouse and zebrafish genomes contain 18, 17 and 24 non-keratin IntFil genes, respectively. Human has 27 of 28 type I “acidic” keratin genes clustered at chromosome (Chr) 17q21.2, and all 26 type II “basic” keratin genes clustered at Chr 12q13.13. Mouse has 27 of 28 type I keratin genes clustered on Chr 11, and all 26 type II clustered on Chr 15. Zebrafish has 18 type I keratin genes scattered on five chromosomes, and 3 type II keratin genes on two chromosomes. Types I and II keratin clusters—reflecting evolutionary blooms of keratin genes along one chromosomal segment—are found in all land animal genomes examined, but not fishes; such rapid gene expansions likely reflect sudden requirements for many novel paralogous proteins having divergent functions to enhance species survival following sea-to-land transition. Using data from the Genotype-Tissue Expression (GTEx) project, tissue-specific keratin expression throughout the human body was reconstructed. Clustering of gene expression patterns revealed similarities in tissue-specific expression patterns for previously described “keratin pairs” (i.e., KRT1/KRT10, KRT8/KRT18, KRT5/KRT14, KRT6/KRT16 and KRT6/KRT17 proteins). The ClinVar database currently lists 26 human disease-causing variants within the various domains of keratin proteins.

The cytoskeleton of the cell is constantly exposed to physical forces that regulate cellular functions. Selected members of the LIM (Lin-11, Isl-1, and Mec-3) domain-containing protein family accumulate along force-bearing actin fibers, with evidence supporting that the LIM domain is solely responsible for this force-induced interaction. However, LIM domain's force-induced interactions are not limited to actin. LIMK1 and LMO1, both containing only two tandem LIM domains, are recruited to force-bearing keratin fibers in epithelial cells. This unique recruitment is mediated by their LIM domains and regulated by the sequences outside the LIM domains. Based on in vitro reconstitution of this interaction, LIMK1 and LMO1 directly interact with stretched keratin 8/18 fibers. These results show that LIM domain's mechano-sensing abilities extend to the keratin cytoskeleton, highlighting the diverse role of LIM proteins in force-regulated signaling. [Display omitted] • LIMK1 and LMO1 proteins selectively and directly bind to force-bearing keratin filaments • This unique recruitment is regulated by the sequences outside the LIM domains • The dynamic nature of LIMK1 and LMO1 binding suggests their role in downstream signaling • The keratin network may serve as a signaling hub for mechano-transduction Kim et al. show that LIMK1 and LMO1, with two tandem LIM domains, directly bind to force-bearing keratin filaments in epithelial cells. The dynamic nature of this unique recruitment suggests their potential involvement in downstream signaling cascades originating from the keratin cytoskeleton, acting as a signaling hub. [ABSTRACT FROM AUTHOR]

Liquid and elastic behaviours of tissues drive their morphology and response to the environment. They appear as the first insight into tissue mechanics. We explore the role of individual cell properties on spheroids of mouse muscle precursor cells and investigate the role of intermediate filaments on surface tension and Young’s modulus. By flattening multicellular myoblast aggregates under magnetic constraint, we measure their rigidity and surface tension and show that they act as highly sensitive macroscopic reporters closely related to microscopic local tension and effective adhesion. Shedding light on the major contributions of acto-myosin contractility, actin organization, and intercellular adhesions, we reveal the role of a major component of intermediate filaments in the muscle, desmin and its organization, on the macroscopic mechanics of these tissue models. Implicated in the mechanical and shape integrity of cells, intermediate filaments are found to be crucial to the mechanics of unorganized muscle tissue models even at an early stage of differentiation both in terms of elasticity and surface tension.

Human epithelial keratin is an intermediate filament protein that serves as a backbone to maintain the stability of the cell nucleus and mechanical stability of the whole cells. The present study focused on two point mutations, F231L and S233L, of the 1B domain of keratin K 1/10 related to the rare genetic skin disease palmoplantar keratoderma (PPK). We used molecular dynamics simulation to study the effects of the mutations on various hierarchical structures, including heterodimers, tetramers, and octamers of the K1/10 1B domain at the atomic scale. The initial results demonstrated that the wild type and mutant proteins were highly similar at the dimer level but had different microstructures and mechanics at a higher-level assembly. A decrease in the hydrophobic interactions and hydrogen bonds at the terminus resulted in weakened mechanical properties of the tetramer and octamer of the F231L mutant. The asymmetrical structure of the S233L tetramer with an uneven distribution of the hydrogen bonds decreased its mechanical properties. However, the S233L mutation provided extra hydrophobic interactions between these mutated amino acid residues in the octamer, leading to improved mechanical properties. The results of the present study provided a deeper understanding of how the differences in point mutations induced the changes in the configuration and mechanical properties at the molecular scale. The differences in these properties may influence keratin assembly at the microscopic scale and ultimately cause diseases at the macroscopic scale. [ABSTRACT FROM AUTHOR]

Here, we describe pathological events potentially involved in the disease pathogenesis of Alexander disease (AxD). This is a primary genetic disorder of astrocyte caused by dominant gain-of-function mutations in the gene coding for an intermediate filament protein glial fibrillary acidic protein (GFAP). Pathologically, this disease is characterized by the upregulation of GFAP and its accumulation as Rosenthal fibers. Although the genetic basis linking GFAP mutations with Alexander disease has been firmly established, the initiating events that promote GFAP accumulation and the role of Rosenthal fibers (RFs) in the disease process remain unknown. Here, we investigate the hypothesis that disease-associated mutations promote GFAP aggregation through aberrant posttranslational modifications. We found high molecular weight GFAP species in the RFs of AxD brains, indicating abnormal GFAP crosslinking as a prominent pathological feature of this disease. In vitro and cell-based studies demonstrate that cystine-generating mutations promote GFAP crosslinking by cysteine-dependent oxidation, resulting in defective GFAP assembly and decreased filament solubility. Moreover, we found GFAP was ubiquitinated in RFs of AxD patients and rodent models, supporting this modification as a critical factor linked to GFAP aggregation. Finally, we found that arginine could increase the solubility of aggregation-prone mutant GFAP by decreasing its ubiquitination and aggregation. Our study suggests a series of pathogenic events leading to AxD, involving interplay between GFAP aggregation and abnormal modifications by GFAP ubiquitination and oxidation. More important, our findings provide a basis for investigating new strategies to treat AxD by targeting abnormal GFAP modifications., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In human hair follicles, the hair‐forming cells express 16 hair keratin genes depending on the differentiation stages. K85 and K35 are the first hair keratins expressed in cortical cells at the early stage of the differentiation. Two types of mutations in the gene encoding K85 are associated with ectodermal dysplasia of hair and nail type. Here, we transfected cultured SW‐13 cells with human K85 and K35 genes and characterized filament formation. The K85–K35 pair formed short filaments in the cytoplasm, which gradually elongated and became thicker and entangled around the nucleus, indicating that K85–K35 promotes lateral association of short intermediate filaments (IFs) into bundles but cannot form IF networks in the cytoplasm. Of the K85 mutations related to ectodermal dysplasia of hair and nail type, a two‐nucleotide (C1448T1449) deletion (delCT) in the protein tail domain of K85 interfered with the K85–K35 filament formation and gave only aggregates, whereas a missense mutation (233A>G) that replaces Arg78 with His (R78H) in the head domain of K85 did not interfere with the filament formation. Transfection of cultured MCF‐7 cells with all the hair keratin gene combinations, K85–K35, K85(R78H)–K35 and K85(delCT)–K35, as well as the individual hair keratin genes, formed well‐developed cytoplasmic IF networks, probably by incorporating into the endogenous cytokeratin IF networks. Thus, the unique de novo assembly properties of the K85–K35 pair might play a key role in the early stage of hair formation.

The intermediate filament protein vimentin plays a key role in cell signaling and stress sensing, as well as an integrator of cytoskeletal dynamics. The vimentin monomer consists of a central rod-like domain and intrinsically disordered head and tail domains. Although the organization of vimentin oligomers in filaments is beginning to be understood, the precise disposition of the tail region remains to be elucidated. Here we observed that electrophilic stress-induced condensation shielded vimentin from recognition by antibodies against specific segments of the tail domain. A detailed characterization revealed that vimentin tail segments are differentially exposed at distinct subcellular locations, both in basal and stress conditions. The 411–423 segment appeared accessible in all cell areas, correlating with vimentin abundance. In contrast, the 419–438 segment was more scantily recognized in perinuclear vimentin and lipoxidative stress-induced bundles, and better detected in peripheral filaments, where it appeared to protrude further from the filament core. These differences persisted in mitotic cells. Interestingly, both tail segments showed closer accessibility in calyculin A-treated cells and phosphomimetic mutants of the C-terminal region. Our results lead us to hypothesize the presence of at least two distinct arrangements of vimentin tail in cells: an “extended” conformation (accessible 419–438 segment), preferentially detected in peripheral areas with looser filaments, and a “packed” conformation (shielded 419–438 segment), preferentially detected at the cell center in robust filaments and lipoxidative stress-induced bundles. These different arrangements could be putatively interconverted by posttranslational modifications, contributing to the versatility of vimentin functions and/or interactions.

While classic cadherin-actin connections in adherens junctions (AJs) have ancient origins, intermediate filament (IF) linkages with desmosomal cadherins arose in vertebrate organisms. In this mini-review, we discuss how overlaying the IF-desmosome network onto the existing cadherin-actin network provided new opportunities to coordinate tissue mechanics with the positioning and function of chemical signaling mediators in the ErbB family of receptor tyrosine kinases. We focus in particular on the complex multi-layered outer covering of the skin, the epidermis, which serves essential barrier and stress sensing/responding functions in terrestrial vertebrates. We will review emerging data showing that desmosome-IF connections, AJ-actin interactions, ErbB family members, and membrane tension are all polarized across the multiple layers of the regenerating epidermis. Importantly, their integration generates differentiation-specific roles in each layer of the epidermis that dictate the form and function of the tissue. In the basal layer, the onset of the differentiation-specific desmosomal cadherin desmoglein 1 (Dsg1) dials down EGFR signaling while working with classic cadherins to remodel cortical actin cytoskeleton and decrease membrane tension to promote cell delamination. In the upper layers, Dsg1 and E-cadherin cooperate to maintain high tension and tune EGFR and ErbB2 activity to create the essential tight junction barrier. Our final outlook discusses the emerging appreciation that the desmosome-IF scaffold not only creates the architecture required for skin’s physical barrier but also creates an immune barrier that keeps inflammation in check.

Mapping intermediate filaments in three dimensions reveals that the organization of these filaments differs across cell types.

Liquid biopsies are promising diagnostic tools for glioma. In this quantitative systematic review, we investigate whether the detection of intermediate filaments (IF) in body fluids can be used as a tool for glioma diagnosis and prognosis. We included all studies in which IF-levels were determined in patients with glioma and healthy controls. Of the 28 identified eligible studies, 12 focussed on levels of GFAP in serum (sGFAP) and were included for metadata analysis. In all studies combined, 62.7% of all grade-IV patients had detectable levels of sGFAP compared to 12.7% of healthy controls. sGFAP did not surpass the limit of detection in lower-grade patients or healthy controls, but sGFAP was significantly elevated in grade-IV glioma (0.12 ng/mL (0.06 − 0.18), P < 0.001) and showed an average median difference of 0.15 ng/mL (0.04 − 0.25, P < 0.01) compared to healthy controls. sGFAP levels were linked to tumour volume, but not to patient outcome. The presence of sGFAP is indicative of grade-IV glioma, but additional studies are necessary to fully determine the usefulness of GFAP in body fluids as a tool for grade-IV glioma diagnosis and follow-up. [ABSTRACT FROM AUTHOR]

Intermediate filament (IntFil) genes arose during early metazoan evolution, to provide mechanical support for plasma membranes contacting/interacting with other cells and the extracellular matrix. Keratin genes comprise the largest subset of IntFil genes. Whereas the first keratin gene appeared in sponge, and three genes in arthropods, more rapid increases in keratin genes occurred in lungfish and amphibian genomes, concomitant with land animal-sea animal divergence (~ 440 to 410 million years ago). Human, mouse and zebrafish genomes contain 18, 17 and 24 non-keratin IntFil genes, respectively. Human has 27 of 28 type I "acidic" keratin genes clustered at chromosome (Chr) 17q21.2, and all 26 type II "basic" keratin genes clustered at Chr 12q13.13. Mouse has 27 of 28 type I keratin genes clustered on Chr 11, and all 26 type II clustered on Chr 15. Zebrafish has 18 type I keratin genes scattered on five chromosomes, and 3 type II keratin genes on two chromosomes. Types I and II keratin clusters—reflecting evolutionary blooms of keratin genes along one chromosomal segment—are found in all land animal genomes examined, but not fishes; such rapid gene expansions likely reflect sudden requirements for many novel paralogous proteins having divergent functions to enhance species survival following sea-to-land transition. Using data from the Genotype-Tissue Expression (GTEx) project, tissue-specific keratin expression throughout the human body was reconstructed. Clustering of gene expression patterns revealed similarities in tissue-specific expression patterns for previously described "keratin pairs" (i.e., KRT1/KRT10, KRT8/KRT18, KRT5/KRT14, KRT6/KRT16 and KRT6/KRT17 proteins). The ClinVar database currently lists 26 human disease-causing variants within the various domains of keratin proteins. [ABSTRACT FROM AUTHOR]

Mechanobiology requires precise quantitative information on processes taking place in specific 3D microenvironments. Connecting the abundance of microscopical, molecular, biochemical, and cell mechanical data with defined topologies has turned out to be extremely difficult. Establishing such structural and functional 3D maps needed for biophysical modeling is a particular challenge for the cytoskeleton, which consists of long and interwoven filamentous polymers coordinating subcellular processes and interactions of cells with their environment. To date, useful tools are available for the segmentation and modeling of actin filaments and microtubules but comprehensive tools for the mapping of intermediate filament organization are still lacking. In this work, we describe a workflow to model and examine the complete 3D arrangement of the keratin intermediate filament cytoskeleton in canine, murine, and human epithelial cells both, in vitro and in vivo. Numerical models are derived from confocal airyscan high-resolution 3D imaging of fluorescence-tagged keratin filaments. They are interrogated and annotated at different length scales using different modes of visualization including immersive virtual reality. In this way, information is provided on network organization at the subcellular level including mesh arrangement, density and isotropic configuration as well as details on filament morphology such as bundling, curvature, and orientation. We show that the comparison of these parameters helps to identify, in quantitative terms, similarities and differences of keratin network organization in epithelial cell types defining subcellular domains, notably basal, apical, lateral, and perinuclear systems. The described approach and the presented data are pivotal for generating mechanobiological models that can be experimentally tested.

The cytoskeleton is a biopolymer network composed of intermediate filaments, actin, and microtubules, which is the main mechanical structure of cells. Vimentin is an intermediate filament protein that regulates the mechanical and contractile properties of cells, thereby reflecting their mechanical properties. In recent years, the "nonmechanical function" of vimentin inside and outside of cells has attracted extensive attention. The content of vimentin in atherosclerotic plaques is increased, and the serum secretion of vimentin in patients with coronary heart disease is remarkably increased. In this review, the mechanistic and nonmechanistic roles of vimentin in atherosclerosis progression were summarized on the basis of current studies. [ABSTRACT FROM AUTHOR]

It has been a long time since researchers have focused on the cytoskeletal proteins' unconventional functions in the nucleus. Subcellular localization of a protein not only affects its functions but also determines the accessibility for cellular processes. Desmin is a muscle-specific, cytoplasmic intermediate filament protein, the cytoplasmic roles of which are defined. Yet, there is some evidence pointing out nuclear functions for desmin. In silico and wet lab analysis shows that desmin can enter and function in the nucleus. Furthermore, the candidate nuclear partners of desmin support the notion that desmin can serve as a transcriptional regulator inside the nucleus. Uncovering the nuclear functions and partners of desmin will provide a new insight into the biological significance of desmin. [ABSTRACT FROM AUTHOR]

Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest), was reported. POLArIS is a fusion protein consisting of a non-immunoglobulin-based recombinant binder Affimer and a green fluorescent protein (GFP), where the Affimer and GFP are rigidly connected to each other. POLArIS probe for molecules of interest can be developed through phage display screening of Affimer. This screening is followed by the rigid connection of fluorescent proteins to the selected Affimers. The Affimer-based POLArIS, however, cannot be used with animal immune libraries for selecting specific binder clones. In addition, multi-color FPM by POLArIS was not available due to the lack of color variations of POLArIS. In this study, we have developed new versions of POLArIS with nanobodies, which are compatible with animal immune libraries, and expanded color variations of POLArIS with cyan/green/yellow/red fluorescent proteins, enabling multi-color orientation imaging for multiple targets. Using nanobody-based POLArIS orientation probes, we performed two-color FPM of F-actin and vimentin in living cells. Furthermore, we made nanobody-based POLArIS probes that have different dipole orientations for adjusting the orientation of fluorescence polarization with respect to the target molecules. These nanobody-based POLArIS with options of colors and dipole orientations will enhance the performance of this probe for broader applications of fluorescence polarization imaging in living cells, tissues, and whole organisms. [Display omitted] • We have developed novel nanobody-based versions of POLArIS, a versatile fluorescent probe for molecular orientation. • We developed color variations of nanobody-based POLArIS, enabling multi-color orientation imaging in living cells. • POLArIS with various dipole orientations enable adjustment of their polarization orientations with respect to their targets. [ABSTRACT FROM AUTHOR]

Background: Desmin is a muscle-specific protein belonging to the intermediate filament family. Desmin mutations are linked to skeletal muscle defects, including inherited myopathies with severe clinical manifestations. The aim of this study was to examine the role of desmin in skeletal muscle remodeling and performance gain induced by muscle mechanical overloading which mimics resistance training.Methods: Plantaris muscles were overloaded by surgical ablation of gastrocnemius and soleus muscles. The functional response of plantaris muscle to mechanical overloading in desmin-deficient mice (DesKO, n = 32) was compared to that of control mice (n = 36) after 7-days or 1-month overloading. To elucidate the molecular mechanisms implicated in the observed partial adaptive response of DesKO muscle, we examined the expression levels of genes involved in muscle growth, myogenesis, inflammation and oxidative energetic metabolism. Moreover, ultrastructure and the proteolysis pathway were explored.Results: Contrary to control, absolute maximal force did not increase in DesKO muscle following 1-month mechanical overloading. Fatigue resistance was also less increased in DesKO as compared to control muscle. Despite impaired functional adaptive response of DesKO mice to mechanical overloading, muscle weight and the number of oxidative MHC2a-positive fibers per cross-section similarly increased in both genotypes after 1-month overloading. However, mechanical overloading-elicited remodeling failed to activate a normal myogenic program after 7-days overloading, resulting in proportionally reduced activation and differentiation of muscle stem cells. Ultrastructural analysis of the plantaris muscle after 1-month overloading revealed muscle fiber damage in DesKO, as indicated by the loss of sarcomere integrity and mitochondrial abnormalities. Moreover, the observed accumulation of autophagosomes and lysosomes in DesKO muscle fibers could indicate a blockage of autophagy. To address this issue, two main proteolysis pathways, the ubiquitin-proteasome system and autophagy, were explored in DesKO and control muscle. Our results suggested an alteration of proteolysis pathways in DesKO muscle in response to mechanical overloading.Conclusion: Taken together, our results show that mechanical overloading increases the negative impact of the lack of desmin on myofibril organization and mitochondria. Furthermore, our results suggest that under these conditions, the repairing activity of autophagy is disturbed. Consequently, force generation is not improved despite muscle growth, suggesting that desmin is required for a complete response to resistance training in skeletal muscle.

Desmin, an intermediate filament protein expressed in muscle cells, plays a key role in the integrity and regulation of the contractile system. Furthermore, the distribution of desmin in cells and its interplay with plasma and organelle membranes are crucial for cell functions; however, the fundamental properties of lipid-desmin interactions remain unknown. Using a water-in-oil method for a limited space system in vitro , we examined the distribution of desmin in three types of phospholipid droplets: 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl- sn -glycero-3-phosphoserine (DOPS). When fluorescent-labeled desmin was observed for 60 min after desmin assembly was initiated by adding 25 mM KCl, desmin accumulated on both the DOPE and DOPS layers; however, it did not accumulate on the DOPC layer of droplets. An increase in salt concentration did not moderate the accumulation. The initial form of either oligomer or mature filament affected the accumulation on each lipid layer. When liposomes were included in the droplets, desmin was associated with DOPE but not on DOPC liposomes. These results suggest that desmin has the potential for association with phospholipids concerning desmin form and lipid shape. The behavior and composition of living membranes may affect the distribution of desmin networks. [Display omitted] • Desmin was associated with DOPE and DOPS layers. • Maturation of desmin filaments weakened the affinity for phospholipid layers. • Desmin could accumulate around hybrid DOPE liposomes. [ABSTRACT FROM AUTHOR]

In human hair follicles, the hair‐forming cells express 16 hair keratin genes depending on the differentiation stages. K85 and K35 are the first hair keratins expressed in cortical cells at the early stage of the differentiation. Two types of mutations in the gene encoding K85 are associated with ectodermal dysplasia of hair and nail type. Here, we transfected cultured SW‐13 cells with human K85 and K35 genes and characterized filament formation. The K85–K35 pair formed short filaments in the cytoplasm, which gradually elongated and became thicker and entangled around the nucleus, indicating that K85–K35 promotes lateral association of short intermediate filaments (IFs) into bundles but cannot form IF networks in the cytoplasm. Of the K85 mutations related to ectodermal dysplasia of hair and nail type, a two‐nucleotide (C1448T1449) deletion (delCT) in the protein tail domain of K85 interfered with the K85–K35 filament formation and gave only aggregates, whereas a missense mutation (233A>G) that replaces Arg78 with His (R78H) in the head domain of K85 did not interfere with the filament formation. Transfection of cultured MCF‐7 cells with all the hair keratin gene combinations, K85–K35, K85(R78H)–K35 and K85(delCT)–K35, as well as the individual hair keratin genes, formed well‐developed cytoplasmic IF networks, probably by incorporating into the endogenous cytokeratin IF networks. Thus, the unique de novo assembly properties of the K85–K35 pair might play a key role in the early stage of hair formation. [ABSTRACT FROM AUTHOR]

Mutations in the type I keratin 16 (Krt16) and its partner type II keratin 6 (Krt6a, Krt6b) cause pachyonychia congenita (PC), a disorder typified by dystrophic nails, painful hyperkeratotic calluses in glabrous skin, and lesions involving other epithelial appendages. The pathophysiology of these symptoms and its relationship to settings in which Krt16 and Krt6 are induced in response to epidermal barrier stress are poorly understood. We report that hyperkeratotic calluses arising in the glabrous skin of individuals with PC and Krt16 null mice share a gene expression signature enriched in genes involved in inflammation and innate immunity, in particular damage-associated molecular patterns. Transcriptional hyper-activation of damage-associated molecular pattern genes occurs following de novo chemical or mechanical irritation to ear skin and in spontaneously arising skin lesions in Krt16 null mice. Genome-wide expression analysis of normal mouse tail skin and benign proliferative lesions reveals a tight, context-dependent coregulation of Krt16 and Krt6 with genes involved in skin barrier maintenance and innate immunity. Our results uncover a role for Krt16 in regulating epithelial inflammation that is relevant to genodermatoses, psoriasis, and cancer and suggest a avenue for the therapeutic management of PC and related disorders.

High levels of the intermediate filament protein keratin 17 (K17) are associated with poor prognoses for several human carcinomas. Studies in mouse models have shown that K17 expression is positively associated with growth, survival, and inflammation in skin and that lack of K17 delays onset of tumorigenesis. K17 occurs in the nucleus of human and mouse tumor keratinocytes where it impacts chromatin architecture, gene expression, and cell proliferation. We report here that K17 is induced following DNA damage and promotes keratinocyte survival. The presence of nuclear K17 is required at an early stage of the double-stranded break (DSB) arm of the DNA damage and repair (DDR) cascade, consistent with its ability to associate with key DDR effectors, including A-H2A.X, 53BP1, and DNA-PKcs. Mice lacking K17 or with attenuated K17 nuclear import showed curtailed initiation in a two-step skin carcinogenesis paradigm. The impact of nuclear-localized K17 on DDR and cell survival provides a basis for the link between K17 induction and poor clinical outcomes for several human carcinomas. [ABSTRACT FROM AUTHOR]

Intermediate filaments (IFs) are key players in multiple cellular processes throughout human tissues. Their biochemical and structural properties are important for understanding filament assembly mechanisms, for interactions between IFs and binding partners, and for developing pharmacological agents that target IFs. IF proteins share a conserved coiled-coil central-rod domain flanked by variable N-terminal 'head' and C-terminal 'tail' domains. There have been several recent advances in our understanding of IF structure from the study of keratins, glial fibrillary acidic protein, and lamin. These include discoveries of (i) a knob–pocket tetramer assembly mechanism in coil 1B; (ii) a lamin-specific coil 1B insert providing a one-half superhelix turn; (iii) helical, yet flexible, linkers within the rod domain; and (iv) the identification of coil 2B residues required for mature filament assembly. Furthermore, the head and tail domains of some IFs contain low-complexity aromatic-rich kinked segments, and structures of IFs with binding partners show electrostatic surfaces are a major contributor to complex formation. These new data advance the connection between IF structure, pathologic mutations, and clinical diseases in humans. Image 1 • Intermediate filament domain structures provide insight into assembly mechanisms. • Heads and tails may contain low-complexity aromatic-rich kinked segments. • Electrostatic surfaces help intermediate filaments bind partner proteins. • The genotype and structurotype contribute to the clinic phenotype in human diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Keratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wild-type K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17-null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization and RNA processing. Key histone modifications and LAP2β (an isoform encoded by TMPO) localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells. [ABSTRACT FROM AUTHOR]

The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14’s codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.

Historically synemin has been studied as an intermediate filament protein. However, synemin also binds the type II regulatory (R) subunit α of protein kinase A (PKA) and protein phosphatase type 2A, thus participating in the PKA and phosphoinositide 3-kinase (PI3K)-Akt and signaling pathways. In addition, recent studies using transgenic mice indicate that a significant function of synemin is its role in signaling pathways in various tissues, including the heart. Recent clinical reports have shown that synemin mutations led to multiple cases of dilated cardiomyopathy. Additionally, a single case of the rare condition ulnar-mammary-like syndrome with left ventricular tachycardia due to a mutation in the synemin gene (SYNM) has been reported. Therefore, this review uses these recent studies to provide a new framework for detailed discussions on synemin tissue distribution, binding partners and synemin in disease. Differences between α- and β-synemin are highlighted. The studies presented here indicate that while synemin does function as an intermediate filament protein, it is unique among this large family of proteins as it is also a regulator of signaling pathways and a crosslinker. Also evident is that the dominant function(s) are isoform-, developmental-, and tissue-specific.

Abstract Background Epidermolytic palmoplantar keratoderma (EPPK) is characterized by hyperkeratotic lesions on palms and soles. The disorder is caused by mutations of keratin 9 (KRT9) or KRT1 gene. Methods Epidermolytic palmoplantar keratoderma was diagnosed by physical examination and histopathological analysis in a five‐generation Chinese family. Mutation was screened by Sanger sequencing. The palmar expression of multiple cytokeratins were analyzed by tape‐stripping and Real‐time PCR. Literatures of EPPK with additional symptoms were reviewed. Results Affected family members showed diffuse palmoplantar keratosis, with knuckle pads, friction‐related lesions and a novel additional symptom of palmar constriction. A heterozygous mutation of c.T491C (p.L164P) of KRT9 was found within the helix initiation motif. The hydrophobic effect was decreased and the initiation of coiled‐coil conformation was delayed. The KRT16/KRT6 expression were significantly increased in the patients, especially on the right, indicating activation of stress‐response and wound‐healing cytokeratins. There were also increased KRT9/KRT2, unchanged KRT10/KRT1, and undetectable KRT14/KRT5 expression. The genetic and phenotypic heterogeneity of EPPK with additional symptoms were summarized by literature review. Conclusion The p.L164P mutation of KRT9 caused EPPK with a novel symptom of palmar constriction. The expression of multiple cytokeratins was altered in EPPK patients.

Although intermediate filaments (IFs) are biochemically and immunologically suggested to exist in plant cells, there are few molecular genetic studies related to the proteins that form these structures. In this study, Arabidopsis AT3G05270 was selected as a candidate gene for a protein constituting IF in plant cells. The protein encoded by AT3G05270 has a large α-helix as well as the IF protein motif indispensable for maintaining the structures of IF. Moreover, fluorescence signals of this protein fused with GFP exhibited cytoskeleton-like filamentous structures in plant cells. Thus, we named the protein encoded by AT3G05270 as Intermediate Filament Motif Protein 1 (IFMoP1). The structures composed of IFMoP1 and their localizations were examined in IFMoP1-GFP-expressing tobacco BY-2 cells whose cell cycle was synchronized using aphidicolin, a DNA synthesis inhibitor, and propyzamide, a microtubule-disrupting agent. The IFMoP1-GFP signals were present at the spindles and phragmoplasts in the mitotic phase. In addition, the frequency of cells with cytoskeleton-like filamentous structures composed of IFMoP1-GFP increased with the increase in cells that completed cell division, and then decreased after several hours. In terms of the relationship in intracellular localization between IFMoP1 and microtubules, the filamentous structures composed of IFMoP1 were present independently of microtubules during interphase. In living cells, these filamentous structures moved along with the nucleus. IFMoP1 co-localized with spindle and phragmoplast microtubules during mitosis, as well as with a part of the cortical microtubules in interphase. [ABSTRACT FROM AUTHOR]

Activated macrophages show increased expression of vimentin, an intermediate filament protein. Macrophages secrete vimentin into extracellular space; however, the functions of extracellular vimentin and the process of vimentin secretion are not clearly defined. We found that oxidized low-density lipoproteins (oxLDL) via CD36 induced vimentin secretion in macrophages. We also revealed that extracellular vimentin induced macrophages to release inflammatory cytokines and augmented oxLDL-induced release of TNF-α and IL-6. Extracellular vimentin activated NF-κB signaling via phosphorylation of focal adhesion kinase (p-FAK) and IκB kinase (p-IκK). Extracellular vimentin also amplified the oxLDL-induced p-IκK increase and IκB decrease. Vimentin-induced TNF-α release was not dependent on Dectin-1, which is known to bind vimentin. We measured serum vimentin concentrations and found that patients with atherosclerotic coronary artery disease had higher levels of serum vimentin than normal subjects. Circulating oxLDL and vimentin concentrations showed a high degree of correlation. In mouse experiments, vimentin concentration was higher in the sera of apoE null mice with western diet–induced atherosclerosis than in the sera of chow diet–fed apoE null mice without atherosclerosis. We concluded that vimentin is secreted by oxLDL/CD36 interaction in macrophages and extracellular vimentin promotes macrophage release of pro-inflammatory cytokines. This may contribute to atherosclerotic inflammation and based on our analysis of serum vimentin, we suggest serum vimentin as a predictive marker for atherosclerosis. Key messages: OxLDL via CD36 induces secretion of vimentin, a cytoskeletal protein in macrophages. Extracellular vimentin induces macrophages to release proinflammatory cytokines such as tumor necrotizing factor-alpha (TNF-α) and this process is mediated by activation of focal adhesion kinase (FAK) and NF-ƙB signaling. Serum concentrations of vimentin in coronary artery disease patients are higher than that in control group. Vimentin concentration is strongly correlated with oxLDL concentration in serum. [ABSTRACT FROM AUTHOR]

Cells are dynamic structures that continually generate and sustain mechanical forces within their environments. Cells respond to mechanical forces by changing their shape, moving, and differentiating. These reactions are caused by intracellular skeletal changes, which induce changes in cellular mechanical properties such as stiffness, elasticity, viscoelasticity, and adhesiveness. Interdisciplinary research combining molecular biology with physics and mechanical engineering has been conducted to characterize cellular mechanical properties and understand the fundamental mechanisms of mechanotransduction. In this review, we focus on the role of cytoskeletal proteins in cellular mechanics. The specific role of each cytoskeletal protein, including actin, intermediate filaments, and microtubules, on cellular elasticity is summarized along with the effects of interactions between the fibers. [ABSTRACT FROM AUTHOR]

This review analyzes data concerning patients with cardiomyopathies or skeletal myopathies associated with a variation in the intermediate filament (IF) synemin gene (SYNM), also referred to as desmuslin (DMN). Molecular studies demonstrate that synemin copolymerizes with desmin and vimentin IF and interacts with vinculin, -actinin, -dystrobrevin, dystrophin, talin, and zyxin. It has been found that synemin is an A-kinase-anchoring protein (AKAP) that anchors protein kinase A (PKA) and modulates the PKA-dependent phosphorylation of several cytoskeletal substrates such as desmin. Because several IF proteins, including desmin, have been implicated in human genetic disorders such as dominant or recessive congenital and adult-onset myopathy, synemin becomes a significant candidate for cardiac and skeletal myopathies of unknown etiology. Because SYNM is a new candidate gene that displays numerous sequence polymorphisms, in this review, we summarize the genetic and clinical literature about SYNM mutations. Protein-changing variants (missense, frameshifts, nonsense) were further evaluated based on structural modifications and amino acid interactions. We present in silico modeling of helical salt-bridges between residues to evaluate the impact of the synemin networks crucial to interactions with cytoskeletal proteins. Finally, a discussion is featured regarding certain variants that may contribute to the disease state. [ABSTRACT FROM AUTHOR]

Desmosome-anchored keratin intermediate filaments (KFs) are essential for epithelial coherence. Yet, desmosomal KF attachment and network organization are still unexplored in vivo. We, therefore, monitored KF network morphogenesis in fluorescent keratin 8 knock-in murine embryos revealing keratin enrichment at newly formed desmosomes followed by KF formation, KF elongation and KF fusion. To examine details of this process and its coupling to desmosome formation, we studied fluorescent keratin and desmosomal protein reporter dynamics in the periphery of expanding HaCaT keratinocyte colonies. Less than 3 min after the start of desmosomal proteins clustering non-filamentous keratin enriched at these sites followed by KF formation and elongation. Subsequently, desmosome-anchored KFs merged into stable bundles generating a rim-and-spokes system consisting of subcortical KFs connecting desmosomes to each other and radial KFs connecting desmosomes to the cytoplasmic KF network. We conclude that desmosomes are organizing centers for the KF cytoskeleton with a hitherto unknown nucleation capacity. [ABSTRACT FROM AUTHOR]

Model organisms are increasingly used to study and understand how neurofilament (NF)‐based neurological diseases develop. However, whether a NF homolog exists in C. elegans remains unclear. We characterize TAG‐63 as a NF‐like protein with sequence homologies to human NEFH carrying various coiled coils as well as clustered phosphorylation sites. TAG‐63 also exhibits features of NFL such as a molecular weight of around 70 kD, the lack of KSP repeats and the ability to form 10 nm filamentous structures in transmission electron micrographs. An anti‐NEFH antibody detects a band at the predicted molecular weight of TAG‐63 in Western blots of whole worm lysates and this band cannot be detected in tag‐63 knockout worms. A transcriptional tag‐63 reporter expresses in a broad range of neurons, and various anti‐NFH antibodies stain worm neurons with an overlapping expression of axonal vesicle transporter UNC‐104(KIF1A). Cultured neurons grow shorter axons when incubating with drugs known to disintegrate the NF network and rhodamine‐labeled in vitro reconstituted TAG‐63 filaments disintegrate upon drug exposure. Speeds of UNC‐104 motors are diminished in tag‐63 mutant worms with visibly increased accumulations of motors along axons. UNC‐104/TAG‐63 and SNB‐1/TAG‐63 not only colocalize in neurons but also revealed positive BiFC (bimolecular fluorescence assay) signals. In summary, we identified and characterized TAG‐63 in C. elegans, and demonstrate that lack of this protein limits axonal transport efficiencies. Additionally, this study would aid in developing NF‐related disease models in the future. [ABSTRACT FROM AUTHOR]

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout (Krt18-KO) or dominant-negative mutant mice (Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force. [ABSTRACT FROM AUTHOR]

The dominant structural feature of intermediate filament (IF) proteins is a centrally located α-helix. These long α-helical segments become paired in a parallel orientation to form coiled-coil dimers. Pairs of dimers further coalesce in an anti-parallel orientation to form tetramers. These early stages of intermediate filament assembly can be accomplished solely by the central α-helices. By contrast, the assembly of tetramers into mature intermediate filaments is reliant upon an N-terminal head domain. IF head domains measure roughly 100 amino acids in length and have long been understood to exist in a state of structural disorder. Here, we describe experiments favoring the unexpected idea that head domains self-associate to form transient structural order in the form of labile cross-β interactions. We propose that this weak form of protein structure allows for dynamic regulation of IF assembly and disassembly. We further offer that what we have learned from studies of IF head domains may represent a simple, unifying template for understanding how thousands of other intrinsically disordered proteins help to establish dynamic morphological order within eukaryotic cells. [ABSTRACT FROM AUTHOR]