Elements Of Cytoskeleton Research Articles

Septin 9 expression regulates 'don't eat me' signals and identifies an immune-epithelial class of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and aggressive liver cancer with limited therapeutic options. Precise classification and immunotherapy are perspectives to improve the treatments. We reported the role of septin 9 in apico-basal polarity and epithelial-to-mesenchymal transition (EMT). Here, we aim to elucidate its role in iCCA. We analyzed single-cell transcriptomes from human iCCA tumor cells based on phenotype and cell state. Knockdown of the septin 9 gene (SEPT9) was done using small interfering RNA (siRNA); interferon-γ (IFN-γ) stimulation was performed using different CCA cells; gene expressions were analyzed by reverse transcription and real-time PCR analysis (RT-qPCR); and immunofluorescence, immunoblotting, and flow cytometry were performed to assess the expression of proteins. The differential distributions of SEPT9 and vimentin (VIM) gene expressions allowed us to define specific cellular trajectories of malignant cells and thus identified distinct clusters of iCCA cells. One cluster was enriched in VIM and extracellular-matrix (ECM) remodeling molecules, and another had high expression of SEPT9 and genes from the 'don't eat me' signal involved in immune escape. This antagonism between SEPT9 and VIM was confirmed by in vitro experiments. Notably, SEPT9 and 'don't eat me' gene expressions were inversely correlated to those of vimentin and the EMT markers. SEPT9 expression was upregulated by IFN-γ and SEPT9 knockdown decreased expression of 'don't eat me' signal genes and increased expression of mesenchymal markers. Cancer Cell Line Encyclopedia (CCLE) transcriptome database analyses confirmed that iCCA cells enriched in septin 9 exhibit epithelial-like features. This study revealed septin 9 as a cytoskeleton element of iCCA epithelial-like cells and a regulator of the immune system response. It also brings new insights into the enigmatic relationship between EMT and immune response. Notably, we decoded a potential mechanism that could sensitize patients to immunotherapies.

Advancing cellular insights: Super-resolution STORM imaging of cytoskeletal structures in human stem and cancer cells

Fluorescence microscopy is an important tool for cell biology and cancer research. Present-day approach of implementing advanced optical microscopy methods combined with immunofluorescence labelling of specific proteins in cells is now able to deliver optical super-resolution up to ∼25 nm. Here we perform super-resolved imaging using standard immunostaining protocol combined with easy stochastic optical reconstruction microscopy (easySTORM) to observe structural differences of two cytoskeleton elements, actin and tubulin in three different cell types namely human bone marrow-derived mesenchymal stem cells (MSCs), human glioblastoma (U87MG) and breast cancer (MDAMB-231) cells. The average width of the actin bundle obtained from STORM images of stem cells is observed to be larger than the same for U87MG and MDAMB-231 cells. No significant difference is however noticed in the width of the tubulin within the same cells. We also study the functional effect on the 2D migration potential of MDAMB-231 cells silenced for NICD1 and β-catenin. Although similar migration speed is observed for cells with the above two conditions compared to their control cells, easySTORM images show that widths of the actin in MDAMB-231 cells in β-catenin silenced is significantly lower than the same in control cells. Such minute differences however are not observable in widefield images. The outcome of our easySTORM investigation should benefit the researchers carrying out detailed investigations of the cellular structure and potential therapeutic applications.

Imaging and Analysis of Drosophila Neural Stem Cell Asymmetric Division.

Cell division is a conserved process among eukaryotes. It is designed to segregate chromosomes into future daughter cells and involves a complex rearrangement of the cytoskeleton, including microtubules and actin filaments. An additional level of complexity is present in asymmetric dividing stem cells because cytoskeleton elements are also regulated by polarity cues. The neural stem cell system of the fruit fly represents a simple model to dissect the mechanisms that control cytoskeleton reorganization during asymmetric division. In this chapter, we propose to describe protocols that allow accurate analysis of microtubule reorganization during cell division in this model.

Abstract 17551: Mapping the Interactome of FLNC in Restrictive Cardiomyopathy

Introduction: Filamin C (FLNC) is an actin crosslinking protein that organizes structural components of the sarcomere and signal transduction complexes. Human mutations in FLNC have been linked to dilated, hypertrophic and restrictive cardiomyopathies. The mechanism connecting mutations in FLNC to sarcomeric impairment remains undefined. We recently reported the ability to model FLNC cardiomyopathy using induced pluripotent stem cell (iPSC) derived engineered heart tissue. Here, we employ a knock-in approach to epitope tag endogenous alleles in iPSC derived cardiomyocytes to explore FLNC genotype-interactome relationships using affinity purification mass spectrometry. Methods: An iPSC line was generated from a patient with restrictive cardiomyopathy caused by an insertion deletion mutation in the ROD2 domain FLNC (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp). CRISPR-Cas9 was used to establish a corrected cell line as a wild type control. 3X-Flag motif was introduced to the N terminus of FLNC using CRISPR-Cas9. iPSCs were then differentiated into cardiomyocytes using standard techniques. Affinity purification mass spectrometry (AP-MS) was performed using the Flag epitope. Interaction data was analyzed using Perseus software. Gene ontology (GO) enrichment analysis was performed using ToppFun. Results: 3X-Flag insertion was confirmed using genomic sequencing. AP-MS identified 281 high confidence interactors of mutant FLNC and 208 interactors of wild type FLNC (FDR 0.01, log2FC >1.5) when compared to IgG controls. Interestingly, 161 proteins were shared between the two genotypes. These were enriched in mitochondrial ribosomal proteins and phosphorylase kinase subunits, potentially revealing an unknown role of FLNC in mitochondria translation and metabolism. Thirty-three proteins were significantly differentially bound to mutant FLNC protein, including tropomyosin 4, caldesmon 1, and filamin A (FDR <0.05, log2FC>1). GO analysis of these proteins revealed enrichment of actin cytoskeleton elements, muscle contraction, and muscle system processes. Conclusions: Human mutations in FLNC lead to disruption of key protein interactions, revealing a potential mechanism for disease pathogenesis in FLNC cardiomyopathy.

Microtubule Regulation in Plants: From Morphological Development to Stress Adaptation.

Microtubules (MTs) are essential elements of the eukaryotic cytoskeleton and are critical for various cell functions. During cell division, plant MTs form highly ordered structures, and cortical MTs guide the cell wall cellulose patterns and thus control cell size and shape. Both are important for morphological development and for adjusting plant growth and plasticity under environmental challenges for stress adaptation. Various MT regulators control the dynamics and organization of MTs in diverse cellular processes and response to developmental and environmental cues. This article summarizes the recent progress in plant MT studies from morphological development to stress responses, discusses the latest techniques applied, and encourages more research into plant MT regulation.

Septins as membrane influencers: direct play or in association with other cytoskeleton partners.

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.

Thermosensing in plants: Deciphering the mechanisms involved in heat sensing and their role in thermoresponse and thermotolerance

With a gradual increase in the ambient temperature plants need to perceive these changes accurately and adjust accordingly in order to adapt and survive. Heat stress affects various developmental stages of plants and adversely impacts yield. Therefore, approaches for developing crops with heat tolerance and high crop productivity have now become major agricultural goals. In the heat sensing process, high temperature causes several changes in various components of the cell, including changes in the membrane fluidity, calcium ions, cytoskeleton elements and protein unfolding. Although some information regarding the signaling mechanisms activated upon sensing of high temperature has been available, only recently new insights have been gained in the thermosensing mechanisms in plants. This review focuses on the various molecular changes that occur within a cell with the rise in temperature and how these are linked to thermomorphogenesis and thermotolerance. Moreover, in the light of upcoming research, the role of photoreceptors, phytohormones, DNA and RNA in heat perception has been given greater attention. • Detrimental effects of heat stress on plants. • Role of various cellular components i.e. cell membranes, proteins, cytoskeleton elements, DNA and RNA in heat perception. • Sensing of high ambient temperature leads to two different responses i.e. thermomorphogenesis and thermotolerance. • Photoreceptor function as thermosensor.

Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood-Brain Barrier In Vitro System.

The mechanisms involved in the interaction of PrP 106-126, a peptide corresponding to the prion protein amyloidogenic region, with the blood–brain barrier (BBB) were studied. PrP 106-126 treatment that was previously shown to impair BBB function, reduced cAMP levels in cultured brain endothelial cells, increased nitric oxide (NO) levels, and changed the activation mode of the small GTPases Rac1 (inactivation) and RhoA (activation). The latter are well established regulators of endothelial barrier properties that act via cytoskeletal elements. Indeed, liquid chromatography-mass spectrometry (LC-MS)-based proteomic profiling study revealed extensive changes in expression of cytoskeleton-related proteins. These results shed light on the nature of the interaction between the prion peptide PrP 106-126 and the BBB and emphasize the importance of the cytoskeleton in endothelium response to prion- induced stress.

Atomic force microscopy identifies the alteration of rheological properties of the cardiac fibroblasts in idiopathic restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare disease characterized by increased ventricular stiffness and preserved ventricular contraction. Various sarcomere gene variants are known to cause RCM; however, more than a half of patients do not harbor such pathogenic variants. We recently demonstrated that cardiac fibroblasts (CFs) play important roles in inhibiting the diastolic function of cardiomyocytes via humoral factors and direct cell–cell contact regardless of sarcomere gene mutations. However, the mechanical properties of CFs that are crucial for intercellular communication and the cardiomyocyte microenvironment remain less understood. In this study, we evaluated the rheological properties of CFs derived from pediatric patients with RCM and healthy control CFs via atomic force microscopy. Then, we estimated the cellular modulus scale factor related to the cell stiffness, fluidity, and Newtonian viscosity of single cells based on the single power-law rheology model and analyzed the comprehensive gene expression profiles via RNA-sequencing. RCM-derived CFs showed significantly higher stiffness and viscosity and lower fluidity compared to healthy control CFs. Furthermore, RNA-sequencing revealed that the signaling pathways associated with cytoskeleton elements were affected in RCM CFs; specifically, cytoskeletal actin-associated genes (ACTN1, ACTA2, and PALLD) were highly expressed in RCM CFs, whereas several tubulin genes (TUBB3, TUBB, TUBA1C, and TUBA1B) were down-regulated. These results implies that the signaling pathways associated with cytoskeletal elements alter the rheological properties of RCM CFs, particularly those related to CF–cardiomyocyte interactions, thereby leading to diastolic cardiac dysfunction in RCM.

Acetylation of α-tubulin on Lys40, a new way to modulate cardiac glucose transport

Cardiovascular disease remains the leading cause of death in the diabetic population. Diabetic patients develop a specific heart condition called diabetic cardiomyopathy that can evolve to heart failure and that is characterized by altered metabolism in the early stages of the disease. Our group previously demonstrated that overfueling the heart with branched-chain amino acids, ketone bodies or fatty acids, substrates that are upregulated in diabetes, inhibits glucose entry in cardiomyocytes through a global rise in protein acetylation that interferes with glucose transporter 4 (GLUT4) translocation to the plasma membrane. However, the acetylated proteins involved in this process were not yet identified. We investigated the role of α-tubulin, an important cytoskeleton element responsible for GLUT4 translocation, that is known to be acetylable on Lys40 (K40). Acetylation level of α-tubulin on K40 was evaluated in a mouse model of diabetes (high-fat diet, 4 months) and in primary cultured adult rat cardiomyocytes. Its acetylation level was modulated by using a pharmacological inhibitor of its deacetylase (tubacin 10 μM, 5 hrs) and by overexpressing a non-acetylable form of α-tubulin (mCherry α-tubulin K40A). GLUT4 translocation was assessed by fluorescent immunostaining in cells expressing the fusion protein HA-GLUT4-GFP. Glucose transport was measured by following the detritiation rate of 2-3H-glucose in insulin-sensitive and insulin-resistant cardiomyocytes. Acetylation of α-tubulin on K40 was significantly enhanced in the heart of diet-induced diabetic mice. Experimentally increasing its acetylation level in cardiomyocytes with tubacin impaired GLUT4 translocation to the plasma membrane and reduced both basal and insulin-dependent glucose uptake. On the other hand, decreasing α-tubulin K40 acetylation via the overexpression of the K40A form of α-tubulin stimulated glucose transport. Interestingly, reducing α-tubulin K40 acetylation similarly promoted glucose entry in insulin-resistant conditions. Cardiac glucose transport can be regulated by modulation of α-tubulin K40 acetylation, which may lead to new therapeutic strategies preventing the metabolic changes occurring during the development of diabetic cardiomyopathy.

Rabies Virus Exploits Cytoskeleton Network to Cause Early Disease Progression and Cellular Dysfunction.

Rabies virus (RABV) is a cunning neurotropic pathogen and causes top priority neglected tropical diseases in the developing world. The genome of RABV consists of nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase L protein (L), respectively. The virus causes neuronal dysfunction instead of neuronal cell death by deregulating the polymerization of the actin and microtubule cytoskeleton and subverts the associated binding and motor proteins for efficient viral progression. These binding proteins mainly maintain neuronal structure, morphology, synaptic integrity, and complex neurophysiological pathways. However, much of the exact mechanism of the viral-cytoskeleton interaction is yet unclear because several binding proteins of the actin-microtubule cytoskeleton are involved in multifaceted pathways to influence the retrograde and anterograde axonal transport of RABV. In this review, all the available scientific results regarding cytoskeleton elements and their possible interactions with RABV have been collected through systematic methodology, and thereby interpreted to explain sneaky features of RABV. The aim is to envisage the pathogenesis of RABV to understand further steps of RABV progression inside the cells. RABV interacts in a number of ways with the cell cytoskeleton to produce degenerative changes in the biochemical and neuropathological trails of neurons and other cell types. Briefly, RABV changes the gene expression of essential cytoskeleton related proteins, depolymerizes actin and microtubules, coordinates the synthesis of inclusion bodies, manipulates microtubules and associated motors proteins, and uses actin for clathrin-mediated entry in different cells. Most importantly, the P is the most intricate protein of RABV that performs complex functions. It artfully operates the dynein motor protein along the tracks of microtubules to assist the replication, transcription, and transport of RABV until its egress from the cell. New remedial insights at subcellular levels are needed to counteract the destabilization of the cytoskeleton under RABV infection to stop its life cycle.

Role of the Cytoskeleton in Steroidogenesis.

Steroidogenesis in the adrenal cortex or gonads is a complicated process modulated by various elements either at the tissue or molecular level. The substrate cholesterol is first delivered to the outer membrane of mitochondria, undergoing a series of enzymatic reactions along with the material exchange between the mitochondria and the ER (endoplasmic reticulum) and ultimately yielding various steroids, such as aldosterone, cortisol, testosterone, and estrone. Several valves are set to adjust the amount of production as per the needs, e.g., StAR (steroidogenic acute regulator) controls the traffic of cholesterol from the outer membrane to the inner membrane of mitochondria which is a rate-limiting step. Moreover, the "need" is partly reflected by trophic signals, like ACTH, LH, and downstream pathways, such as the intracellular cAMP pathway, representing the endocrinal regulation of steroid synthesis. The coordinated activities of these related factors are all associated with another crucial cellular constituent, the cytoskeleton, which plays a crucial role in cellular architecture and substrate trafficking. Though considerable studies have been performed regarding steroid synthesis, details regarding the upstream signaling pathways and mechanisms of the regulation by the cytoskeleton network still remain unclear. The metabolism and interplays of the pivotal cellular organelles with cytoskeleton are worth exploring as well. This review summarizes the research of different periods, describing the roles of specific cytoskeleton elements in steroidogenesis and related signaling pathways involved in steroid synthesis. In addition, we discuss the inner cytoskeletal network involved in steroidogenic processes, such as mitochondrial movement, organelle interactions, and cholesterol trafficking.

ВЛИЯНИЕ МОДЕЛИРУЕМОЙ МИКРОГРАВИТАЦИИ НА СОДЕРЖАНИЕ мРНК ГЕНОВ, КОДИРУЮЩИХ ЦИТОСКЕЛЕТНЫЕ БЕЛКИ, И АЦЕТИЛИРОВАНИЕ ГИСТОНОВ В ЯИЧНИКАХ DROSOPHILA MELANOGASTER

Maintenance of the human reproductive health is vital in context of preserving living species in remote space missions. Changes in the gene expression due to microgravity can lead to degradation of fertility; however, the mechanism that triggers these changes is not fully understood. In our investigation we determined mRNA levels in the prime cytoskeletal proteins and analyzed histone methylation and acetylation activities in Drosophila melanogaster ovaries during 79-hr microgravity modeled with the use of a random positioning machine (RPM), and per os introduction of essential phospholipids. The results showed that the fruit flies exposure to modeled microgravity over the entire oogenesis cycle increased levels of mRNA genes encoding the cytoskeletal proteins in oocytes and that this can be, probably, associated with increased acetylation of the histone H3 Lys9 and Lys27. Essential phospholipids seem to prevent growth and/or initiate mRNA reductions in the majority of cytoskeleton elements in the flies exposed to modeled microgravity; however, the tubulin and lamin B binding proteins increased in the respective controls. Essential phospholipids leveled histone 3 Lys9 acetylation in modeled microgravity but enhanced Lys27 acetylation in the control.

Morphological and ultrastructural characterization of neurospheres spontaneously generated in the culture from sheep ovarian cortical cells.

Neurospheres (NS) derived from adult stem cells of non-neural tissues represent a promising source of neural stem cells (NSCs) and neural progenitor cells (NPCs) for autologous cell therapy. Knowing the fine structure of NS cells is essential for characterizing them during differentiation or oncogenic transformation. NS are generated by culturing ovarian cortical cells (OCCs) under specific conditions. To establish whether these OCCs exhibited a similar morphophenotype as those from the central nervous system (CNS) reported in the literature, sheep OCCs were cultured for 21 days to generate NS. Expression levels of pluripotency (Nanog, octamer-binding transcription factor 4 [Oct4], and SRY-box transcription factor 2 [Sox2]) and NSCs/NPCs (nestin, paired box 6 [Pax6], and p75 neurotrophin receptor [P75NTR]) transcripts were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the NSC/NPC antigens were immunolocalized, and structural and ultrastructural analyses were performed in OCC-NS on Days 10, 15, and 21 in culture. Spheroids expressed transcripts and antigens of pluripotency as well as NSCs/NPCs. Cells were arranged into an inner core, with frequent apoptotic and degenerative events, and a peripheral epithelial-like cover with abundant cytoplasmic organelles, apical microvilli, and filament bundles of cytoskeleton elements. Adherens junctions and apical tight and lateral loose interdigitations were found in peripheral cells that eventually lost apical-basal polarization, which might indicate their disengaging/aggregating from/to the NS. We can conclude that OCC-NS shares the most structural and ultrastructural characteristics with CNS-NS.

Finite element analysis of the influence of cyclic strain on cells anchored to substrates with varying properties.

The response of cytoskeleton to mechanical cues plays a pivotal role in understanding several aspects of cellular growth, migration, and cell-cell and cell-matrix interactions under normal and diseased conditions. Finite element analysis (FEA) has become a powerful computational technique to study the response of cytoskeleton in the maintenance of overall cellular mechanics. With the revelation of role of external mechanical microenvironment on cell mechanics, FEA models have also been developed to simulate the effect of substrate stiffness on the mechanical properties of cancer cells. However, the models developed so far model cellular response under static mode, whereas in physiological condition, cells always experience dynamic loading conditions. To develop a more accurate model of cell-extracellular matrix (ECM) interactions, this paper models the cytoskeleton and other parts of the cell by beam and solid elements respectively, assuming spherical morphology of the cell. The stiffness and roughness of extracellular matrix were varied. Furthermore, static and dynamic sinusoidal loads were applied through a flat plate indenter on the cell along with providing sinusoidal strain at the substrate. It is observed that due to axial loading, cell reaches a plastic region, and when the sinusoidal loading is added to the axial load, the cell experiences permanent deformation. Degradation of the cytoskeleton elements and a physiologically more relevant spherical cap shape of the cell were also considered during the analysis. This study suggests that asperity topology of the substrate and indirect cyclic load can play a significant role in the shape alterations and motion of a cell.

Microtubules as Major Regulators of Endothelial Function: Implication for Lung Injury.

Endothelial dysfunction has been attributed as one of the major complications in COVID-19 patients, a global pandemic that has already caused over 4 million deaths worldwide. The dysfunction of endothelial barrier is characterized by an increase in endothelial permeability and inflammatory responses, and has even broader implications in the pathogenesis of acute respiratory syndromes such as ARDS, sepsis and chronic illnesses represented by pulmonary arterial hypertension and interstitial lung disease. The structural integrity of endothelial barrier is maintained by cytoskeleton elements, cell-substrate focal adhesion and adhesive cell junctions. Agonist-mediated changes in endothelial permeability are directly associated with reorganization of actomyosin cytoskeleton leading to cell contraction and opening of intercellular gaps or enhancement of cortical actin cytoskeleton associated with strengthening of endothelial barrier. The role of actin cytoskeleton remodeling in endothelial barrier regulation has taken the central stage, but the impact of microtubules in this process remains less explored and under-appreciated. This review will summarize the current knowledge on the crosstalk between microtubules dynamics and actin cytoskeleton remodeling, describe the signaling mechanisms mediating this crosstalk, discuss epigenetic regulation of microtubules stability and its nexus with endothelial barrier maintenance, and overview a role of microtubules in targeted delivery of signaling molecules regulating endothelial permeability and inflammation.

Cytoskeleton Reorganization in EndMT-The Role in Cancer and Fibrotic Diseases.

Chronic inflammation promotes endothelial plasticity, leading to the development of several diseases, including fibrosis and cancer in numerous organs. The basis of those processes is a phenomenon called the endothelial–mesenchymal transition (EndMT), which results in the delamination of tightly connected endothelial cells that acquire a mesenchymal phenotype. EndMT-derived cells, known as the myofibroblasts or cancer-associated fibroblasts (CAFs), are characterized by the loss of cell–cell junctions, loss of endothelial markers, and gain in mesenchymal ones. As a result, the endothelium ceases its primary ability to maintain patent and functional capillaries and induce new blood vessels. At the same time, it acquires the migration and invasion potential typical of mesenchymal cells. The observed modulation of cell shape, increasedcell movement, and invasion abilities are connected with cytoskeleton reorganization. This paper focuses on the review of current knowledge about the molecular pathways involved in the modulation of each cytoskeleton element (microfilaments, microtubule, and intermediate filaments) during EndMT and their role as the potential targets for cancer and fibrosis treatment.

Cytoskeleton and Associated Proteins: Pleiotropic JNK Substrates and Regulators.

This review extensively reports data from the literature concerning the complex relationships between the stress-induced c-Jun N-terminal kinases (JNKs) and the four main cytoskeleton elements, which are actin filaments, microtubules, intermediate filaments, and septins. To a lesser extent, we also focused on the two membrane-associated cytoskeletons spectrin and ESCRT-III. We gather the mechanisms controlling cytoskeleton-associated JNK activation and the known cytoskeleton-related substrates directly phosphorylated by JNK. We also point out specific locations of the JNK upstream regulators at cytoskeletal components. We finally compile available techniques and tools that could allow a better characterization of the interplay between the different types of cytoskeleton filaments upon JNK-mediated stress and during development. This overview may bring new important information for applied medical research.

Molecular response of a sub-antarctic population of the blue mussel (Mytilus edulis platensis) to a moderate thermal stress

The Kerguelen Islands (49°26′S, 69°50′E) represent a unique environment due to their geographical isolation, which protects them from anthropogenic pollution. The ability of the endemic mussel, part of the Mytilus complex, to cope with moderate heat stress was explored using omic tools. Transcripts involved in six major metabolic functions were selected and the qRT-PCR data indicated mainly changes in aerobic and anaerobic energy metabolism and stress response. Proteomic comparisons revealed a typical stress response pattern with cytoskeleton modifications and elements suggesting increased energy metabolism. Results also suggest conservation of protein homeostasis by the long-lasting presence of HSP while a general decrease in transcription is observed. The overall findings are consistent with an adaptive response to moderate stresses in mussels in good physiological condition, i.e. living in a low-impact site, and with the literature concerning this model species. Therefore, local blue mussels could be advantageously integrated into biomonitoring strategies, especially in the context of Global Change.

The Cytoskeletal Elements MAP2 and NF-L Show Substantial Alterations in Different Stroke Models While Elevated Serum Levels Highlight Especially MAP2 as a Sensitive Biomarker in Stroke Patients

In the setting of ischemic stroke, the neurofilament subunit NF-L and the microtubule-associated protein MAP2 have proven to be exceptionally ischemia-sensitive elements of the neuronal cytoskeleton. Since alterations of the cytoskeleton have been linked to the transition from reversible to irreversible tissue damage, the present study investigates underlying time- and region-specific alterations of NF-L and MAP2 in different animal models of focal cerebral ischemia. Although NF-L is increasingly established as a clinical stroke biomarker, MAP2 serum measurements after stroke are still lacking. Therefore, the present study further compares serum levels of MAP2 with NF-L in stroke patients. In the applied animal models, MAP2-related immunofluorescence intensities were decreased in ischemic areas, whereas the abundance of NF-L degradation products accounted for an increase of NF-L-related immunofluorescence intensity. Accordingly, Western blot analyses of ischemic areas revealed decreased protein levels of both MAP2 and NF-L. The cytoskeletal alterations are further reflected at an ultrastructural level as indicated by a significant reduction of detectable neurofilaments in cortical axons of ischemia-affected areas. Moreover, atomic force microscopy measurements confirmed altered mechanical properties as indicated by a decreased elastic strength in ischemia-affected tissue. In addition to the results from the animal models, stroke patients exhibited significantly elevated serum levels of MAP2, which increased with infarct size, whereas serum levels of NF-L did not differ significantly. Thus, MAP2 appears to be a more sensitive stroke biomarker than NF-L, especially for early neuronal damage. This perspective is strengthened by the results from the animal models, showing MAP2-related alterations at earlier time points compared to NF-L. The profound ischemia-induced alterations further qualify both cytoskeletal elements as promising targets for neuroprotective therapies.