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Polyploid giant cancer cells (PGCCs) are a commonly observed histological feature of human tumors and are particularly prominent in late stage and drug resistant cancers. The chromosomal duplication conferred by their aneuploidy gives rise to DNA damage resistance and complex tumor cell karyotypes, a driving factor in chemotherapy resistance and disease relapse. Furthermore, PGCCs also exhibit key cytoskeletal features that give rise to a distinct biophysical phenotype, including increased density of polymerized actin and vimentin intermediate filaments, nuclear and cytoskeletal stiffening, increased traction force, and migratory persistence. Despite recent research highlighting the role PGCCs play in cancer progression, this population of tumor cells remains poorly characterized in terms of their biophysical properties. In this review, we will discuss the various aspects of their biomolecular phenotype, such as increased stemness as well as a mixed EMT signature. These features have been extensively associated with tumorigenesis and recurrence, and aggressive cancers. Additionally, we will also examine the distinct PGCC cytoskeletal features of actin and filamentous vimentin. Specifically, how the differential organization of these networks serve to support their increased size and drive migratory persistence. These findings could shed light on potential therapeutic strategies that allow for specific elimination or mitigation of the invasive potential of these polyploid cancer cells. Lastly, we will examine how the biophysical and molecular phenotype of PGCCs combine to tip the scale in favor of promoting cancer progression, presenting an important target in the clinical treatment of cancer.

BACKGROUND: Actin stress fibers are abundant in cultured cells, but little is known about them in vivo. In podocytes, much evidence suggests that mechanobiologic mechanisms underlie podocyte shape and adhesion in health and in injury, with structural changes to actin stress fibers potentially responsible for pathologic changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. A technology to image the actin cytoskeleton at high resolution is needed to better understand the role of structures such as actin stress fibers in podocytes. METHODS: We developed the first visualization technique capable of resolving the three-dimensional cytoskeletal network in mouse podocytes in detail, while definitively identifying the proteins that comprise this network. This technique integrates membrane extraction, focused ion-beam scanning electron microscopy, and machine learning image segmentation. RESULTS: Using isolated mouse glomeruli from healthy animals, we observed actin cables and intermediate filaments linking the interdigitated podocyte foot processes to newly described contractile actin structures, located at the periphery of the podocyte cell body. Actin cables within foot processes formed a continuous, mesh-like, electron-dense sheet that incorporated the slit diaphragms. CONCLUSIONS: Our new technique revealed, for the first time, the detailed three-dimensional organization of actin networks in healthy podocytes. In addition to being consistent with the gel compression hypothesis, which posits that foot processes connected by slit diaphragms act together to counterbalance the hydrodynamic forces across the glomerular filtration barrier, our data provide insight into how podocytes respond to mechanical cues from their surrounding environment.

Cells form networks in animal tissues through synaptic, chemical and adhesive links. Invertebrate muscle cells often connect to other cells through desmosomes, adhesive junctions anchored by intermediate filaments. To study desmosomal networks, we skeletonised 853 muscle cells and their desmosomal partners in volume electron microscopy data covering an entire larva of the annelidPlatynereis. Muscle cells adhere to each other, to epithelial, glial, ciliated, and bristle-producing cells and to the basal lamina, forming a desmosomal connectome of over 2,000 cells. The aciculae – chitin rods that form an endoskeleton in the segmental appendages – are highly connected hubs in this network. This agrees with the many degrees of freedom of their movement, as revealed by video microscopy. Mapping motoneuron synapses to the desmosomal connectome allowed us to infer the extent of tissue influenced by motoneurons. Our work shows how cellular-level maps of synaptic and adherent force networks can elucidate body mechanics.

Nuclear lamins are ancient type V intermediate filaments with diverse functions that include maintaining nuclear shape, mechanosignaling, tethering and stabilizing chromatin, regulating gene expression, and contributing to cell cycle progression. Despite these numerous roles, an outstanding question has been how lamins are regulated. Accumulating work indicates that a range of lamin post-translational modifications (PTMs) control their functions both in homeostatic cells and in disease states such as progeria, muscular dystrophy, and viral infection. Here, we review the current knowledge of the diverse types of PTMs that regulate lamins in a site-specific manner. We highlight methods that can be used to characterize lamina PTMs whose functions are currently unknown and provide a perspective on the future of the lamin PTM field.

Super-resolution microscopy revealed that the plant nuclear lamina proteins NMCP1 and NMCP2 organize into filamentous networks with filament bundles., Plant genomes lack genes encoding intermediate filament proteins, including lamins; however, functional lamin analogues are presumed to exist in plants. Plant-specific coiled-coil proteins, that is, nuclear matrix constituent proteins (NMCPs), are the most likely candidates as the structural elements of the nuclear lamina because they exhibit a lamin-like domain arrangement. They are exclusively localized at the nuclear periphery and have functions that are analogous to those of lamins. However, their assembly into filamentous polymers has not yet been confirmed. In this study, we examined the higher-order structure of NMCP1 and NMCP2 in Apium graveolens cells by using stimulated emission depletion microscopy combined with immunofluorescence cell labelling. Our analyses revealed that NMCP1 and NMCP2 form intricate filamentous networks, which include thick segments consisting of filament bundles, forming a dense filamentous layer extending across the nuclear periphery. Furthermore, the outermost chromatin distribution was found to be in the nucleoplasm-facing region of the nuclear lamina. Recombinant Daucus carota NMCP1 with a His-tag produced in Escherichia coli refolded into dimers and self-assembled into filaments and filament bundles. These results suggest that NMCP1 and NMCP2 organize into the nuclear lamina by forming a filamentous network with filament bundles that localize at the nuclear periphery.

Objectives The human adult vocal fold mucosa, especially, superficial layer of the lamina propria (Reinke's space) has attracted notice as an important vibrating structure. However, fine structures of the stratified squamous epithelium of the human adult vocal fold, which is another histological component of the mucosa, remain enigmatic. Methods Three normal human adult vocal folds and epiglottises and three newborn vocal folds were investigated. Observations using transmission electron microscopy and light microscopy including immunohistochemistry were performed. Results The most obvious feature of the epithelium of the human adult vocal folds was that the intercellular spaces between adjacent epithelial cells were large (984 ± 186 nm) compared with the stratified squamous epithelium of the human adult epiglottis and the human newborn vocal fold. Even though intercellular spaces between adjacent epithelial cells of the human adult vocal fold were large, desmosomes at the junctions of two adjacent epithelial cells made firm intercellular adhesion. Tonofilaments composed of the bundles of intermediate filaments anchored to the desmosomes. Desmosomes formed a continuous cytoskeletal network throughout the epithelial cells and epithelium of the human adult vocal fold. In addition, a great deal of E-cadherin (adhesive glycoprotein) was present between epithelial cells especially the lower half of the stratified squamous epithelium of the human adult vocal fold. Conclusions From the functional morphological point of view, stratified squamous epithelium of the human adult vocal fold seems to form a structural framework of tensile strength with pliability suitable structure for vibration.

Arterial smooth muscle cells (ASMCs), the predominant cell type within the arterial wall, detect and respond to external mechanical forces. These forces can be derived from blood flow (i.e. pressure and stretch) or from the supporting extracellular matrix (i.e. stiffness and topography). The healthy arterial wall is elastic, allowing the artery to change shape in response to changes in blood pressure, a property known as arterial compliance. As we age, the mechanical forces applied to ASMCs change; blood pressure and arterial wall rigidity increase and result in a reduction in arterial compliance. These changes in mechanical environment enhance ASMC contractility and promote disease-associated changes in ASMC phenotype. For mechanical stimuli to programme ASMCs, forces must influence the cell’s load-bearing apparatus, the cytoskeleton. Comprised of an interconnected network of actin filaments, microtubules and intermediate filaments, each cytoskeletal component has distinct mechanical properties that enable ASMCs to respond to changes within the mechanical environment whilst maintaining cell integrity. In this review, we discuss how mechanically driven cytoskeletal reorganisation programmes ASMC function and phenotypic switching.

Peripherin (PRPH), a type III intermediate filament, assembles with neurofilaments in neurons of the peripheral nervous system, including lower motor neurons (LMN). To evaluate the role of PRPH in LMN degeneration, we assessed PRPH and neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and serum of 91 patients with motor neuron diseases (MND) and 69 controls. Overall, we found PRPH to be more concentrated in serum than in CSF. Serum PRPH resulted significantly increased in MND patients but it was unrelated to CSF‐NfL or survival in the amyotrophic lateral sclerosis (ALS) subset. PRPH might represent a marker of LMN involvement.

Although activation of hepatic stellate cells (HSCs) plays a central role in the development of liver fibrosis, the mechanism underlying the activation of HSCs remains unclear. Keratin 17 (KRT17), a member of the intermediate filament family, can regulate tumor cell proliferation and migration. The current study aimed to elucidate the role of KRT17 in the activation of HSCs and the mechanisms underlying liver fibrosis.The expression of KRT17 was determined using immunohistochemistry in tissue microarray. Western blotting and qRT-PCR assays were used to determine the KRT17 expression in fibrotic liver tissues obtained from human subjects and mice. LX-2 cells were treated with TGF-β1 recombinant protein and adipocyte differentiation mixture (MDI) mix to induce and reverse LX-2 cell activation, respectively, in order to explore the correlation between KRT17 and HSC activation. Additionally, cell proliferation and migration abilities of LX-2 cells transfected with KRT17-overexpressing plasmid or small interfering RNA were determined using CCK-8, flow cytometry, Transwell, and wound healing assays. Finally, rescue assay was used to explore the role of KRT17 in HSC activation and epithelial-mesenchymal transition (EMT).The expression of KRT17 was higher in the human and mouse fibrotic liver tissues than in healthy liver tissues, and it was positively correlated with HSC activation. Upregulated KRT17 enhanced proliferation, migration, HSC activation and EMT in LX-2 cells, while knockdown of KRT17 reversed these effects. TGF-β1 recombinant protein accelerated KRT17-mediated EMT, HSC activation and proliferation, while TGF-β1 inhibitor counteracted the effect of KRT17KRT17 promoted HSC activation, proliferation and EMT in hepatic fibrosis probably via TGF-β1 signaling, and KRT17 might serve as a therapeutic target for the treatment of liver fibrosis.

Gliosis is a complex process comprising upregulation of intermediate filament (IF) proteins, particularly glial fibrillary acidic protein (GFAP) and vimentin, changes in glial cell morphology (hypertrophy) and increased deposition of inhibitory extracellular matrix molecules. Gliosis is common to numerous pathologies and can have deleterious effects on tissue function and regeneration. The role of IFs in gliosis is controversial, but a key hypothesized function is the stabilization of glial cell hypertrophy. Here, we developed RNAi approaches to examine the role of GFAP and vimentin in vivo in a murine model of inherited retinal degeneration, the Rhodopsin knockout (Rho-/- ) mouse. Specifically, we sought to examine the role of these IFs in the establishment of Muller glial hypertrophy during progressive degeneration, as opposed to (more commonly assessed) acute injury. Prevention of Gfap upregulation had a significant effect on the morphology of reactive Muller glia cells in vivo and, more strikingly, the reduction of Vimentin expression almost completely prevented these cells from undergoing degeneration-associated hypertrophy. Moreover, and in contrast to studies in knockout mice, simultaneous suppression of both GFAP and vimentin expression led to severe changes in the cytoarchitecture of the retina, in both diseased and wild-type eyes. These data demonstrate a crucial role for Vimentin, as well as GFAP, in the establishment of glial hypertrophy and support the further exploration of RNAi-mediated knockdown of vimentin as a potential therapeutic approach for modulating scar formation in the degenerating retina.

Background Desmin is the major intermediate filament (IF) protein in human heart and skeletal muscle. So-called ‘desminopathies’ are disorders due to pathogenic variants in the DES gene and are associated with skeletal myopathies and/or various types of cardiomyopathies. So far, only a limited number of DES pathogenic variants have been identified and functionally characterized. Methods and results Using a Sanger- and next generation sequencing (NGS) approach in patients with various types of cardiomyopathies, we identified two novel, non-synonymous missense DES variants: p.(Ile402Thr) and p.(Glu410Lys). Mutation carriers developed dilated (DCM) or arrhythmogenic cardiomyopathy (ACM), and cardiac conduction disease, leading to spare out the exercise-induced polymorphic ventricular tachycardia; we moved this variant to data in brief. To investigate the functional impact of these four DES variants, transfection experiments using SW-13 and H9c2 cells with native and mutant desmin were performed and filament assembly was analyzed by confocal microscopy. The DES_p.(Ile402Thr) and DES_p.(Glu410Lys) cells showed filament assembly defects forming cytoplasmic desmin aggregates. Furthermore, immunohistochemical and ultrastructural analysis of myocardial tissue from mutation carriers with the DES_p.(Glu410Lys) pathogenic variant supported the in vitro results. Conclusions Our in vitro results supported the classification of DES_p.(Ile402Thr) and DES_p.(Glu410Lys) as novel pathogenic variants and demonstrated that the cardiac phenotypes associated with DES variants are diverse and cell culture experiments improve in silico analysis and genetic counseling because the pathogenicity of a variant can be clarified.

The beak or rhamphotheca in turtles is a horny lamina that replaces the teeth. Its origin, development, structure, and protein composition are here presented. At mid-development stages, the epidermis of the maxilla and mandible gives rise to placodes that enlarge and merge into laminae through an intense cell proliferation. In these expanding laminae, the epidermis gives rise to 5-8 layers of embryonic epidermis where coarse filaments accumulate for the initial keratinization of cells destined to be sloughed before hatching. Underneath the embryonic epidermis of the beak numerous layers of spindle-shaped beta-cells are produced while they are absent in other skin regions. Beta-cells contain hard corneous material and give rise to the corneous layer of the beak whose external layers desquamate due to wearing and mechanical abrasion. Beta-catenin is present in nuclei of proliferating keratinocytes of the germinal layer likely responding to a wnt signal, but also is part of the adhesive junctions located among beak keratinocytes. The thick corneous layer is made of mature corneocytes connected one to another along their irregular perimeter by an unknown cementing material and junctional remnants. Immunolabeling shows that the main components of the horny beak are Corneous Beta Proteins (CBPs) of 10-15 kDa which genes are located in the Epidermal Differentiation Complex (EDC) of the turtle genome. Specific CBPs, in addition to a lower amount of Intermediate Filament Keratins, accumulate in the horny beak. Compaction of the main proteins with other unknown, minor proteins give rise to the hard corneous material of the beak.

Background Alexander disease (AxD) is a rare neurodegenerative disorder that is caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament that is primarily expressed by astrocytes. In AxD, mutant GFAP in combination with increased GFAP expression result in astrocyte dysfunction and the accumulation of Rosenthal fibers. A neuroinflammatory environment consisting primarily of macrophage lineage cells has been observed in AxD patients and mouse models. Methods To examine if macrophage lineage cells could serve as a therapeutic target in AxD, GFAP knock-in mutant AxD model mice were treated with a colony-stimulating factor 1 receptor (CSF1R) inhibitor, pexidartinib. The effects of pexidartinib treatment on disease phenotypes were assessed. Results In AxD model mice, pexidartinib administration depleted macrophages in the CNS and caused elevation of GFAP transcript and protein levels with minimal impacts on other phenotypes including body weight, stress response activation, chemokine/cytokine expression, and T cell infiltration. Conclusions Together, these results highlight the complicated role that macrophages can play in neurological diseases and do not support the use of pexidartinib as a therapy for AxD.

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in intestinal epithelial cells (IEC; PleΔIEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal α6β4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the PleΔIEC colon.

Astrocytes regulate synaptic communication and are essential for proper brain functioning. In Alzheimer's disease (AD) astrocytes become reactive, which is characterized by an increased expression of intermediate filament proteins and cellular hypertrophy. Reactive astrocytes are found in close association with amyloid‐beta (Aβ) deposits. Synaptic communication and neuronal network function could be directly modulated by reactive astrocytes, potentially contributing to cognitive decline in AD. In this review, we focus on reactive astrocytes as treatment targets in AD in the APPswePS1dE9 AD mouse model, a widely used model to study amyloidosis and gliosis. We first give an overview of the model; that is, how it was generated, which cells express the transgenes, and the effect of its genetic background on Aβ pathology. Subsequently, to determine whether modifying reactive astrocytes in AD could influence pathogenesis and cognition, we review studies using this mouse model in which interventions were directly targeted at reactive astrocytes or had an indirect effect on reactive astrocytes. Overall, studies specifically targeting astrocytes to reduce astrogliosis showed beneficial effects on cognition, which indicates that targeting astrocytes should be included in developing novel therapies for AD., MAIN POINTS The APPswePS1dE9 mouse displays extensive amyloid‐induced reactive astrogliosis.Reactive astrocytes play a pivotal role in AD pathogenesis as they affect synaptic function.Targeting reactive astrocytes may help to prevent cognitive decline.

The process of autophagy plays an important role in the life of the cell, in particular, in the process of maintaining proteostasis. In muscle cells, the process of autophagy is highly dynamic due to the need for constant renewal of the degrading proteins of the Z-disk region during muscle contraction. In this case, the cytoskeleton and, in particular, intermediate filaments play a major role in maintaining the structural and functional integrity of the muscle cell. We hypothesize that disruption of the structure of intermediate filaments of muscle cells, in particular, desmin, leads to a change in the dynamics of the process of autophagy. We have demonstrated that, in C2C12 cells, a mutation of the desmin gene (DES L345P) leads to a significant increase in the baseline level of autophagy and the rate of degradation of cellular components due to the process of autophagy. It has been shown that, in cells with the mutation DES L345P, after stimulation of the process of autophagy by serum starvation for 2 and 4 h, the amount of LC3-II protein is halved relative to cells with wild-type desmin. The rate of conversion of LC3-I to LC3-II remains unchanged. This observation may be associated with an increase in the base level of the process of autophagy and the rate of degradation of autophagosomes for the removal of mutant aggregate forms of the desmin protein from the cell due to mutation L345P.

Lamins are nucleoskeletal proteins of mammalian cells that stabilize the structure and maintain the rigidity of the nucleus. These type V intermediate filament proteins which are predominantly of A and B types provide necessary tensile strength to the nucleus. Single amino acid missense mutations occurring all over the lamin A protein form a cluster of human diseases termed as laminopathies, most of which principally affect the muscle and cardiac tissues responsible for load bearing functionalities of the body. One such mutation is A350P which causes dilated cardiomyopathy in patients. It is postulated that a change from alanine to proline in the α-helical coiled-coil forming 2B rod domain of the protein might severely disrupt the propensity of the filaments to polymerise into functional higher order structures required to form a fully functional lamina with its characteristic elasticity. In this study, we have elucidated for the very first time, the application of active microrheology employing oscillating optical tweezers to investigate any alterations in the viscoelastic parameters of the mutant protein meshwork in vitro, which might translate into possible changes in nuclear plasticity. We confirmed our findings from this robust yet fast method by imaging both the wild type and mutant lamin A networks using a super resolution microscope, and observed changes in the mesh size which corroborate our measured changes in the viscoelastic parameters of the lamins. This method could thus be extended to conduct microrheological measurements on any intermediate filament protein thus bearing significant implications in laminopathies and other diseases associated with intermediate filaments.

Cells in the maculae flavae (MFe) are inferred to be involved in the metabolism of extracellular matrices of the human vocal fold mucosa. The latest research has supported the hypothesis that the tension caused by phonation (vocal fold vibration) regulates the behavior of these cells in the MFe of the human vocal fold. Tensile and compressive strains have direct effects on cell morphology and structure including changes in cytoskeletal structure and organization. Cytoskeletons are one of the structures which play a role as mechanoreceptors for the cells. The microstructure of the intermediate filaments and the expression of their proteins were investigated regarding the cells in the MFe of the human vocal fold unphonated over a decade. The adult vocal fold mucosa of a 64-year-old male with cerebral hemorrhage unphonated for 11 years was investigated by immunohistochemistry and electron microscopy. The intermediate filaments in the cytoplasm of the cells had become fewer in number. And the expression of their characteristic proteins (vimentin, desmin, GFAP) was also reduced. The results of this study are consistent with the hypothesis that mechanotransduction caused by vocal fold vibration could possibly be a factor in regulating the function and fate of the cells in the MFe.

The ability of cells to move through small spaces depends on the mechanical properties of the cellular cytoskeleton and on nuclear deformability. In mammalian cells, the cytoskeleton is comprised of three interacting, semi-flexible polymer networks: actin, microtubules, and intermediate filaments (IF). Recent experiments of mouse embryonic fibroblasts with and without vimentin have shown that the IF vimentin plays a role in confined cell motility. We, therefore, develop a minimal model of cells moving through confined geometries that effectively includes all three types of cytoskeletal filaments with a cell consisting of an actomyosin cortex and a deformable cell nucleus and mechanical connections between the two cortices the outer actomyosin one and the inner nuclear one. By decreasing the amount of vimentin, we find that the cell speed is typically faster for vimentin-null cells as compared to cells with vimentin. Vimentin-null cells also contain more deformed nuclei in confinement. Finally, vimentin affects nucleus positioning within the cell. By positing that as the nucleus position deviates further from the center of mass of the cell, microtubules become more oriented in a particular direction to enhance cell persistence or polarity, we show that vimentin-nulls are more persistent than vimentin-full cells. The enhanced persistence indicates that the vimentin-null cells are more subjugated by the confinement since their internal polarization mechanism that depends on cross-talk of the centrosome with the nucleus and other cytoskeletal connections is diminished. In other words, the vimentin-null cells rely more heavily on external cues. Our modeling results present a quantitative interpretation for recent experiments and have implications for understanding the role of vimentin in the epithelial-mesenchymal transition., Comment: 16 pages, 13 figures

Plectin, a high-molecular-mass cytolinker, is abundantly expressed in the central nervous system (CNS). Currently, a limited amount of data about plectin in the CNS prevents us from seeing the complete picture of how plectin affects the functioning of the CNS as a whole. Yet, by analogy to its role in other tissues, it is anticipated that, in the CNS, plectin also functions as the key cytoskeleton interlinking molecule. Thus, it is likely involved in signalling processes, thereby affecting numerous fundamental functions in the brain and spinal cord. Versatile direct and indirect interactions of plectin with cytoskeletal filaments and enzymes in the cells of the CNS in normal physiological and in pathologic conditions remain to be fully addressed. Several pathologies of the CNS related to plectin have been discovered in patients with plectinopathies. However, in view of plectin as an integrator of a cohesive mesh of cellular proteins, it is important that the role of plectin is also considered in other CNS pathologies. This review summarizes the current knowledge of plectin in the CNS, focusing on plectin isoforms that have been detected in the CNS, along with its expression profile and distribution alongside diverse cytoskeleton filaments in CNS cell types. Considering that the bidirectional communication between neurons and glial cells, especially astrocytes, is crucial for proper functioning of the CNS, we place particular emphasis on the known roles of plectin in neurons, and we propose possible roles of plectin in astrocytes.

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.

Mesenchymal stem cells hold great promise as a therapeutic cell source for a variety of diseases such as osteoarthritis and degenerative disc disease. Their unique homing ability and multipotentiality play key roles in their applications in medicine. Specifically, hMSCs capability to adhere and migrate to damaged tissue's extracellular matrix is essential for serving their therapeutic purpose. The roles of some cytoskeletal elements, such as actin and microtubules, in cell migration and adhesion have been well described, while that of intermediate filaments (IFs) is not well understood. There is increasing evidence that vimentin IFs also function in cell spreading and adhesion, but their relationships in MSCs have not been fully characterized. In this work, we sought to understand how vimentin IFs govern adhesion and cellular cytoplasmic projection formation in human MSCs. Using shRNA to knockdown vimentin IFs in MSCs, we found that vimentin IFs are necessary for the cellular structural integrity required for cell-substrate adhesion and for forming and maintaining cellular projections.

Intermediate filaments (IFs) perform a diverse set of well-known functions including providing structural support for the cell and resistance to mechanical stress, yet recent evidence has revealed unexpected roles for IFs as stress response proteins. Previously, it was shown that the type III IF protein vimentin forms cage-like structures around centrosome-associated proteins destined for degradation, structures referred to as aggresomes, suggesting a role for vimentin in protein turnover. However, vimentin's function at the aggresome has remained largely understudied. In a recent report, vimentin was shown to be dispensable for aggresome formation, but played a critical role in protein turnover at the aggresome through localizing proteostasis-related machineries, such as proteasomes, to the aggresome. Here, we review evidence for vimentin's function in proteostasis and highlight the organismal implications of these findings.

Maturation of the corneous material of feathers, scutate scales, claws and beak is a special case of hard cornification since it mainly derives from the accumulation of small feather corneous beta proteins (FCBPs) of 9–12 kDa with a central beta-pleated sheet region, formerly indicated as feather beta keratins. FCBPs contain a relatively high amount of cysteines that likely form numerous –S-S- in the corneous material of these skin appendages. The present immunocytochemical study shows that sulfhydryl oxidase and FCBPs are associated in the differentiating keratinocytes and corneous layers of the epidermis, scales, claws, beak and barb-barbule cells during chick development. The enzyme appears localized in pre-corneous and corneous layers and in differentiating barb-barbule cells where it likely determines formation of -S-S- bonds. This maturation transformation completed in corneous layers of scales, beak, claws and feathers determines increase of hardness and mechanical resistance that, in feathers, is needed for protection and sustaining flight. The process of cornification in chick skin appendages and feathers is discussed in relation to the general process of formation of hard corneous material in vertebrate skin appendages. This occurs by the association of intermediate filament proteins (IFKs, formerly indicates as alpha-keratins) and keratin-associated proteins (KAPs) or CBPs.

Here, we review the development, morphology, genes, and proteins of claws in reptiles. Claws likely form owing to the inductive influence of phalangeal mesenchyme on the apical epidermis of developing digits, resulting in hyperproliferation and intense protein synthesis in the dorsal epidermis, which forms the unguis. The tip of claws results from prevalent cell proliferation and distal movement along most of the ungueal epidermis in comparison to the ventral surface forming the subunguis. Asymmetrical growth between the unguis and subunguis forces beta-cells from the unguis to rotate into the apical part of the subunguis, sharpening the claw tip. Further sharpening occurs by scratching and mechanical wearing. Ungueal keratinocytes elongate, form an intricate perimeter and cementing junctions, and remain united impeding desquamation. In contrast, thin keratinocytes in the subunguis form a smooth perimeter, accumulate less corneous beta proteins (CBPs) and cysteine-poor intermediate filament (IF)-keratins, and desquamate. In addition to prevalent glycine-cysteine-tyrosine rich CBPs, special cysteine-rich IF-keratins are also synthesized in the claw, generating numerous SS bonds that harden the thick and compact corneous material. Desquamation and mechanical wear at the tip ensure that the unguis curvature remains approximately stable over time. Reptilian claws are likely very ancient in evolution, although the unguis differentiated like the outer scale surface of scales, while the subunguis might have derived from the inner scale surface. The few hair-like IF-keratins synthesized in reptilian claws indicate that ancestors of sauropsids and mammals shared cysteine-rich IF-keratins. However, the number of these keratins remained low in reptiles, while new types of CBPs function to strengthen claws.

Polyploidal giant cancer cells (PGCCs) are multinucleated chemoresistant cancer cells found in heterogeneous solid tumors. Due in part to their apparent dormancy, the effect of PGCCs on cancer progression has remained largely unstudied. Recent studies have highlighted the critical role of PGCCs as aggressive and chemoresistant cancer cells, as well as their ability to undergo amitotic budding to escape dormancy. Our recent study demonstrated the unique biophysical properties of PGCCs, as well as their unusual migratory persistence. Here we unveil the critical function of vimentin intermediate filaments (VIFs) in maintaining the structural integrity of PGCCs and enhancing their migratory persistence. We performed in-depth single-cell analysis to examine the distribution of VIFs and their role in migratory persistence. We found that PGCCs rely heavily on their uniquely distributed and polarized VIF network to enhance their transition from a jammed to an unjammed state to allow for directional migration. Both the inhibition of VIFs with acrylamide and small interfering RNA knockdown of vimentin significantly decreased PGCC migration and resulted in a loss of PGCC volume. Because PGCCs rely on their VIF network to direct migration and to maintain their enlarged morphology, targeting vimentin or vimentin cross-linking proteins could provide a therapeutic approach to mitigate the impact of these chemoresistant cells in cancer progression and to improve patient outcomes with chemotherapy.

The major constituent of the nuclear envelope is the nuclear lamina. A fibrous meshwork of lamin filaments spanning underneath the nuclear membrane provides mechanical support to the nucleus. Lamins, type V intermediate filament proteins, are also involved in many other nuclear activities such as DNA repair and transcription. Recent structural studies provide new insight into how lamins assemble into ∼3.5nm thick filaments, which is in contrast with cytoplasmic intermediate filaments. The thinnest component of the cell cytoskeleton exhibits surprising mechanical properties. Here, we critically review and discuss structural and mechanical aspects of single lamin filaments.

Purpose To analyze the co-expression of the intermediate filaments GFAP and cytokeratin in 326 pituitary adenomas with regard to the distribution pattern, the subtype of the adenoma and clinical prognostic data. Methods Tissue from 326 pituitary adenomas and 13 normal anterior pituitaries collected in the Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, between 2006 and 2009 was investigated by immunohistochemistry, immunofluorescence and electron microscopy. Results Co-expression of intermediate filaments GFAP and cytokeratin was associated with hormone expression in 62/278 cases (22%), but only found in 2/48 (4%) of null cell adenomas (p Conclusions Cellular co-expression of the intermediate filaments GFAP and cytokeratin in pituitary adenomas and the pituitary was demonstrated and shown to be associated with hormone expression and low recurrence rate. The results are discussed with regard to the biology of folliculostellate cells, neural transformation and tumor stem cells. This study may complement the understanding of pituitary adenoma biology.

Введение. Несмотря на определенный прогресс в изучении патоморфологических механизмов сепсиса и усовершенствовании методов терапии критических состояний, летальность при сепсисе сохраняется на высоком уровне. При тяжелом системном воспалении нарушается морфофункциональное состояние кожного покрова, однако обусловливающие этот эффект механизмы описаны поверхностно. Нуждается в уточнении роль изменения экспрессии генов кератиноцитов цитоскелета, приводящая к нарушению их дифференцировки и миграции на фоне действия эндогенного фактора, индуцированного тяжелым системным воспалением. Цель работы - оценка молекулярно-генетического профиля дифференцировки и миграции кератиноцитов на фоне системного воспаления. Методика. Фрагменты интактной кожи пациентов с подтвержденным сепсисом (n=46) были изучены методом полимеразной цепной реакции в режиме реального времени для определения экспрессии генов промежуточных филаментов кератиноцитов KRT1, KRT10, KRT5, KRT14 и KRT16. Результаты. У пациентов в условии системного воспаления относительная экспрессия KRT1, KRT10, KRT5, KRT14 и KRT16 в интактной коже снижена, несмотря на сохранение уровня экспрессии генов, продукты которых поддерживают агрегацию филаментов и стабильность цитоскелета - FLG, IVL. Заключение. В интактной коже на фоне системного воспаления в отсутствие внешнего повреждающего фактора отмечается разобщение дифференцировки и пролиферации кератиноцитов, способное привести к нарушению барьерной функции кожи. Introduction. Despite some progress in studying pathomorphological mechanisms of sepsis and improvement of therapy for critical conditions, mortality in sepsis remains high. In severe systemic inflammation, the morpho-functional state of skin is compromised; however, the mechanisms responsible for this effect are not completely understood. The role of mutations in keratinocyte cytoskeletal genes leading to disorders of keratinocyte differentiation and migration under the action of an endogenous factor induced by severe systemic inflammation, needs to be clarified. The aim of this work was to assess the molecular genetic profile of keratinocyte differentiation and migration under systemic inflammation. Methods. Fragments of intact skin from patients with confirmed sepsis (n=46) were studied with real-time polymerase chain reaction to determine the expression of keratin intermediate filament KRT1, KRT10, KRT5, KRT14, and KRT16 genes. Results. In patients with systemic inflammation, the relative expression of KRT1, KRT10, KRT5, KRT14, and KRT16 in intact skin was reduced, despite the normal expression of genes whose products support filament aggregation and cytoskeletal stability (FLG and IVL). Conclusion. In the intact skin under systemic inflammation in the absence of an external damaging factor, uncoupling of the keratinocyte differentiation from the keratinocyte proliferation was observed, which may lead to dysfunction of the skin barrier.

Axonal cytoskeletal components of neuron have been studied and modeled by using different approaches. The axonal cytoskeleton comprises of microtubules (MTs), which mostly control the overall property and behavior of axon. However, MTs contain crosslinks which are MT associated proteins (mainly tau proteins), intermediate filaments (mainly neurofilaments or NFs in case of brain axon), and microfilaments (MFs). Due to the micrometer level length scale of the components, experimental approaches such as microscopy are inconvenient. In this regard, several computational approaches, such as fully atomistic molecular dynamics (MD) simulation, coarse grained (CG) simulation, or finite element analysis (FEA) can be considered reasonable approaches for observing the behavior of the cytoskeletal components and determining their mechanical properties. Among the cytoskeletal components, MTs and MFs are well studied, but the behavior of taus and NFs are not studied comprehensively. Over the last two decades, the computational approaches have been improved manifold to determine and analyze numerous aspects of cytoskeletal components. Due to structurally disordered nature of tau and NF we lack sufficient literature on these components, almost all the structural and behavioral aspects have been analyzed in depth for MT and MF – for which computational studies have played vital role. This study attempts to discuss the current scenario of these computational approaches performed on cytoskeletal components, as well as recent advancements. It attempts to benchmark the current capability of computational approaches to find out properties, behavior, and dynamics of cytoskeletal components, which is required to further advance using similar maneuvers. Importantly, we also pinpoint the possibilities of future studies through which the current limitations in the tau and NF territories can be effectively addressed.

The development of the beak in turtles is poorly known. Beak development has been analyzed by immunofluorescent methods for studying cell proliferation and localization of specific proteins. The flat two-layered epidermis covering the turtle embryo at mid stage of development becomes columnar in the oral region and is associated with an increase of mesenchymal density as in placodes. Using 5BrdU, an intense cell proliferation is observed in the oral and epidermal cells covering the maxilla and mandibular bones, probably stimulated by the underlying mesenchyme in continuation with maxillary and mandibular bones. Expansion and fusion of these placodes give rise to the corneous beak. Beta catenin, mainly junctional but also sparsely detected in nuclei of the germinal layer of the beak epithelium, maintains united the differentiating keratinocytes that form a stratified corneous epithelium. This is initially composed of some layers of large cells that accumulate intermediate filament keratins (IFKs) and give rise to a keratinized embryonic epidermis destined to slough around hatching. The following corneocytes accumulate IFKs but mainly type I/II corneous beta proteins (CBPs) and form a corneous beak. These CBPs appear present with lower intensity in the beak than in the shell, but the higher intensity obtained with a general antibody against CBPs indicates that other CBPs contribute to the composition and stiffness of beak corneous material. The egg-tooth in continuation with the stratum corneum of the maxillary beak develops from a localized proliferation and comprises a thick embryonic epidermis accumulating IFKs under which large beta-cells connected by adhesion proteins accumulate CBPs contributing to hardening of this temporary organ.

Aim To study the effect of mechanical stress on the cytoskeleton in lens epithelial cells following conventional phacoemulsification surgery (CPS) and femtosecond laser-assisted cataract surgery (FLACS). Methods The cytoskeleton of the epithelial cells of the anterior lens capsules (ALC) removed by CPS and FLACS was examined by immunohistochemistry. Expression of the intermediate filament, glial fibrillary acidic protein (GFAP), and glutamine synthetase (GS) immunoreactivity were detected. In order to map the actin network of cells, fluorescently labeled phalloidin was used. The samples were examined using confocal laser scanning microscopy. Results GFAP expression was visible in a larger number of the epithelial cells after CPS compared to FLACS. In CPS sample's epithelial cells, GFAP immunoreactivity indicated robust morphological change. Regarding the actin filaments, the presence of tubular elements connecting epithelial cells, regular actin pattern and marked cortical network after CPS were found. Following FLACS, the actin cytoskeleton of the epithelial cells remained densely structured, and the tubular elements were undetectable, however, the above-mentioned regular actin pattern and the marked cortical network were visible. Conclusion The conventional removal of the ALC induces more robust changes of the cytoskeleton of the lens epithelial cells.

Aims Plectin, a universally expressed multi-functional cytolinker protein, is crucial for intermediate filament networking, including crosstalk with actomyosin and microtubules. In addition to its involvement in a number of diseases affecting skin, skeletal muscle, heart, and other stress-exposed tissues, indications for a neuropathological role of plectin have emerged. Having identified P1c as the major isoform expressed in neural tissues in previous studies, our aim for the present work was to investigate whether, and by which mechanism(s), the targeted deletion of this isoform affects neuritogenesis and proper nerve cell functioning. Methods For ex vivo phenotyping, we used dorsal root ganglion and hippocampal neurons derived from isoform P1c-deficient and plectin-null mice, complemented by in vitro experiments using purified proteins and cell fractions. To assess the physiological significance of the phenotypic alterations observed in P1c-deficient neurons, P1c-deficient and wild-type littermate mice were subjected to standard behavioural tests. Results We demonstrate that P1c affects axonal microtubule dynamics by isoform-specific interaction with tubulin. P1c deficiency in neurons leads to altered dynamics of microtubules and excessive association with tau protein, affecting neuritogenesis, neurite branching, growth cone morphology, and translocation and directionality of movement of vesicles and mitochondria. On the organismal level, we found P1c deficiency manifesting as impaired pain sensitivity, diminished learning capabilities and reduced long-term memory of mice. Conclusions Revealing a regulatory role of plectin scaffolds in microtubule-dependent nerve cell functions, our results have potential implications for cytoskeleton-related neuropathies.

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.

Neurofilaments are proteins selectively expressed in the cytoskeleton of neurons, and increased levels are a marker of damage. Elevated neurofilament levels can serve as a marker of ongoing disease activity as well as a tool to measure response to therapeutic intervention. The potential utility of neurofilaments has drastically increased as recent advances have made it possible to measure levels in both the cerebrospinal fluid and blood. There is mounting evidence that neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (NfH) are abnormal in a host of neurodegenerative diseases. In this review we examine how both of these proteins behave across diseases and what we know about how these biomarkers relate to in vivo white matter pathology and each other.

Peri-infarct depolarization (PID), one kind of spreading depolarization, contributes to infarct volume enlargement after ischemic stroke. Astrocytes participate in PIDs by various mechanisms. The roles of glial fibrillary acidic protein (GFAP) and vimentin (Vim), intermediate filament proteins in astrocytes, however, in PIDs induction and propagation remain unknown. Middle cerebral artery occlusion (MCAO) model was made in 9 GFAP−/−Vim−/− and 9 wild-type (WT) C57BL/6 mice. Using 4-wavelength optical intrinsic signal imaging (OIS), we identified PIDs as consistent, red and blue interaction waves in the cortical reflectance that slowly propagated peripherally from the origin site. Five propagation patterns of PIDs were observed after MCAO in mice, namely, latero-medial, medial-lateral, rostro-caudal, caudo-rostral, and collision. Additionally, the frequency, propagation velocity, and duration of PIDs between GFAP−/−Vim−/− and WT mice were not significantly different (p > 0.05). Furthermore, no significant difference was found in infarct volume and brain edema between the two groups. In conclusion, the 4-wavelength OIS system allows acquisition of high temporal-spatial resolution color images for analyzing temporal-spatial characteristics of PIDs in detail. GFAP and Vim in astrocytes are not involved in PIDs after MCAO in mice.

The nuclear lamina is a meshwork of intermediate filament proteins, and lamin A is the primary mechanical protein. An altered splicing of lamin A, known as progerin, causes the disease Hutchinson-Gilford progeria syndrome. Progerin-expressing cells have altered nuclear shapes and stiffened nuclear lamina with microaggregates of progerin. Here, progerin microaggregate inclusions in the lamina are shown to lead to cellular and multicellular dysfunction. We show with Comsol simulations that stiffened inclusions causes redistribution of normally homogeneous forces, and this redistribution is dependent on the stiffness difference and relatively independent of inclusion size. We also show mechanotransmission changes associated with progerin expression in cells under confinement and cells under external forces. Endothelial cells expressing progerin do not align properly with patterning. Fibroblasts expressing progerin do not align properly to applied cyclic force. Combined, these studies show that altered nuclear lamina mechanics and microstructure impacts cytoskeletal force transmission through the cell.

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.

In this study, we established the components of the buffalo spermatogonial stem cell (SSC) niche in prenatal (38.5-280 days of gestation), neonatal, prepubertal, and adult testes histochemically and immunohistochemically. Immunohistochemically, the changes in vimentin expression were evaluated. During prenatal life, the niche of primordial germ cells was formed by the surrounding mesenchymal cells and fibroblasts. The basement membrane (BM) was not organized at this stage. At 7.6 cm crown-rump length, testicular cords with a thin BM were seen. At the late gestational period, testicular cords with well-organized BM were seen. At the neonatal stage, the testicular cords were lined with distinct BM, while during the prepubertal period, distinct BM with peritubular myoid cells (PMCs) were seen. In adult testes, the seminiferous tubules were surrounded by a thick BM surrounded by PMCs. PM cells were only single-layered as seen in H&E-stained paraffin sections, picrosirius red-stained, and Masson?s trichrome-stained paraffin sections. Collagen fibers were seen to increase quantitatively to provide strength and cushioning to the developing niche of spermatogonial stem cells in the testes. The expression of vimentin increased to a certain extent until adult life, indicating its significant role as an intermediate filament during development.

Apoptosis plays a key role in removing abnormal or senescent cells, maintaining the overall health of the tissue, and coordinating individual development. Recently, it has been discovered that the intracellular cytoskeleton plays a role in the apoptotic process. In addition, the regulatory role of extracellular matrix (ECM) fibrous proteins, which can be considered as the extracellular skeleton, in the process of apoptosis is rarely summarized. In this review, we collect the latest knowledge about how fibrous proteins inside and outside cells regulate apoptosis. We describe how ECM fibrous proteins participate in the regulation of death receptor and mitochondrial pathways through various signaling cascades mediated by integrins. We then explore the molecular mechanisms by which intracellular intermediate filaments regulate cell apoptosis by regulating death receptors on the cell membrane surface. Similarly, we report on novel supporting functions of microtubules in the execution phase of apoptosis and discuss their formation mechanisms. Finally, we discuss that the polypeptide fragments formed by caspase degradation of ECM fibrous proteins and intracellular intermediate filament act as local regulatory signals to play different regulatory roles in apoptosis.

INTRODUCTION 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. MATERIALS AND METHODS We included all studies in which IF-levels were determined in patients with glioma and healthy controls. Of the 28 identified eligible studies, 12 focused on levels of GFAP in serum (sGFAP) and were included for metadata analysis. RESULTS 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. CONCLUSION 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.

Epidermolysis bullosa simplex (EBS) is a blistering dermatosis that is mostly caused by dominant mutations in KRT5 and KRT14. In this study, we investigated one patient with localized recessive EBS caused by novel homozygous c.1474T > C mutations in KRT5. Biochemical experiments showed a mutation-induced alteration in the keratin 5 structure, intraepidermal blisters, and collapsed keratin intermediate filaments, but no quantitative change at the protein levels and interaction between keratin 5 and keratin 14. Moreover, we found that MAPK signaling was inhibited, while desmosomal protein desmoglein 1 (DSG1) was upregulated upon KRT5 mutation. Inhibition of EGFR phosphorylation upregulated DSG1 levels in an in vitro model. Collectively, our findings suggest that this mutation leads to localized recessive EBS and that keratin 5 is involved in maintaining DSG1 via activating MAPK signaling.

Desmosomes, strong cell–cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise five major protein classes – desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin – whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with fluorescence recovery after photobleaching (FRAP) and fluorescence loss and localisation after photobleaching (FLAP), we show that desmosomes consist of two contrasting protein moieties or modules: a very stable moiety of desmosomal cadherins, desmoplakin and plakoglobin, and a highly mobile plakophilin (Pkp2a). As desmosomes mature from Ca2+ dependence to Ca2+-independent hyper-adhesion, their stability increases, but Pkp2a remains highly mobile. We show that desmosome downregulation during growth-factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable moiety and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in desmosomes. This article has an associated First Person interview with the first author of the paper.