Are the Head and Tail Domains of Intermediate Filaments Really Unstructured Regions?

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

Vimentin filaments integrate low-complexity domains in a complex helical structure.

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion-beam milling, cryo-electron microscopy and tomography to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, intertwined and flexible helical structure of 40 α-helices in cross-section, organized into five protofibrils. Surprisingly, the intrinsically disordered head domains form a fiber in the lumen of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking mechanical strength and stretchability.

Correlation analysis between smoke exposure and serum neurofilament light chain in adults: a cross-sectional study.

BACKGROUND: Smoke exposure is a prevalent and well-documented risk factor for various diseases across different organ systems. Serum neurofilament light chain (sNfL) has emerged as a promising biomarker for a multitude of nervous system disorders. However, there is a notable paucity of research exploring the associations between smoke exposure and sNfL levels. METHODS: We conducted a comprehensive analysis of the National Health and Nutrition Examination Survey (NHANES) cross-sectional data spanning the years 2013 to 2014. Serum cotinine levels were classified into the following three groups: < 0.05, 0.05-2.99, and ≥ 3 ng/ml. Multiple linear regression models were employed to assess the relationships between serum cotinine levels and sNfL levels. Additionally, we utilized restricted cubic spline analyses to elucidate the potential nonlinear relationship between serum cotinine and sNfL levels. RESULTS: A total of 2053 participants were included in our present research. Among these individuals, the mean age was 47.04 ± 15.32 years, and males accounted for 48.2% of the total study population. After adjusting the full model, serum cotinine was positively correlated with sNfl in the second group (ß = 0.08, 95%CI 0.01-0.15) and in the highest concentration of serum cotinine (ß = 0.10, 95%CI 0.01-0.19) compared to the group with the lowest serum cotinine concentrations. Current smokers, in comparison to non-smokers, exhibited a trend toward elevated sNfL levels (ß = 0.07, 95%CI 0.01-0.13). Furthermore, subgroup analyses revealed interactions between serum cotinine levels and different age groups (P for interaction = 0.001) and gender stratification (P for interaction = 0.015) on sNfL levels. CONCLUSION: The study suggested that serum cotinine was significantly and positively associated with sNfl levels in adult participants. Furthermore, current smokers tend to exhibit elevated sNfL levels. This research sheds light on the potential implications of smoke exposure on neurological function impairment and underscores the importance of further exploration in this area.

The redox-responsive roles of intermediate filaments in cellular stress detection, integration and mitigation.

Intermediate filaments are critical for cell and tissue homeostasis and for stress responses. Cytoplasmic intermediate filaments form versatile and dynamic assemblies that interconnect cellular organelles, participate in signaling and protect cells and tissues against stress. Here we have focused on their involvement in redox signaling and oxidative stress, which arises in numerous pathophysiological situations. We pay special attention to type III intermediate filaments, mainly vimentin, because it provides a physical interface for redox signaling, stress responses and mechanosensing. Vimentin possesses a single cysteine residue that is a target for multiple oxidants and electrophiles. This conserved residue fine tunes vimentin assembly, response to oxidative stress and crosstalk with other cellular structures. Here we integrate evidence from the intermediate filament and redox biology fields to propose intermediate filaments as redox sentinel networks of the cell. To support this, we appraise how vimentin detects and orchestrates cellular responses to oxidative and electrophilic stress.

Transmit and protect: The mechanical functions of intermediate filaments.

New aspects of the unique mechanical properties of intermediate filaments (IFs) continue to emerge from studies that illuminate the structure and mechanical response of single filaments, the interaction of intermediate filaments with each other or with other cytoskeletal elements, and the viscoelasticity of the networks that these intermediate filaments form. The relation of purified IF network mechanics to the role of IFs in cells and tissues is a particularly active area, with several new demonstrations of the unique and essential role that intermediate filament networks play in determining the mechanical response of biological materials, especially to large deformations, and the mechanisms by which intermediate filaments protect the nucleus from mechanical stresses that cells and tissues encounter in vivo.

How do disordered head domains assist in the assembly of intermediate filaments?

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

A Comparison of Two Analytical Approaches for the Quantification of Neurofilament Light Chain, a Biomarker of Axonal Damage in Multiple Sclerosis.

Neurofilament light chain (NfL), is a neuron-specific cytoskeletal protein detected in extracellular fluid following axonal damage. Extensive research has focused on NfL quantification in CSF, establishing it as a prognostic biomarker of disability progression in Multiple Sclerosis (MS). Our study used a new commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kit and Single Molecular Array (Simoa) advanced technology to assess serum NfL levels in MS patients and Healthy Controls (HC). Verifying the most accurate, cost-effective methodology will benefit its application in clinical settings. Blood samples were collected from 54 MS patients and 30 HC. Protocols accompanying the kits were followed. The ELISA thershold was set as 3 S.D. above the mean of the HC. For Simoa, the Z-score calculation created by Jens Kuhle's group was applied (with permission). Samples exceeding the threshold or z-score ≥1.5 indicated subclinical disease activity. To our knowledge, this is the first study to find strong-positive correlation between ELISA and Simoa for the quantification of NfL in serum (r = 0.919). Despite the strong correlation, Simoa has better analytical sensitivity and can detect small changes in samples making it valuable in clinical settings. Further research is required to evaluate whether serum NfL quantification using ELISA could be utilized to predict disability progression.

Exploring the Potential of Metal-Based Candidate Drugs as Modulators of the Cytoskeleton.

During recent years, accumulating evidence suggested that metal-based candidate drugs are promising modulators of cytoskeletal and cytoskeleton-associated proteins. This was substantiated by the identification and validation of actin, vimentin and plectin as targets of distinct ruthenium(II)- and platinum(II)-based modulators. Despite this, structural information about molecular interaction is scarcely available. Here, we compile the scattered reports about metal-based candidate molecules that influence the cytoskeleton, its associated proteins and explore their potential to interfere in cancer-related processes, including proliferation, invasion and the epithelial-to-mesenchymal transition. Advances in this field depend crucially on determining binding sites and on gaining comprehensive insight into molecular drug-target interactions. These are key steps towards establishing yet elusive structure-activity relationships.

Cryo-EM Structures of Chronic Traumatic Encephalopathy Tau Filaments with PET Ligand Flortaucipir.

Positron emission tomography (PET) imaging allows monitoring the progression of amyloid aggregation in the living brain. [18F]-Flortaucipir is the only approved PET tracer compound for the visualisation of tau aggregation. Here, we describe cryo-EM experiments on tau filaments in the presence and absence of flortaucipir. We used tau filaments isolated from the brain of an individual with Alzheimer's disease (AD), and from the brain of an individual with primary age-related tauopathy (PART) with a co-pathology of chronic traumatic encephalopathy (CTE). Unexpectedly, we were unable to visualise additional cryo-EM density for flortaucipir for AD paired helical or straight filaments (PHFs or SFs), but we did observe density for flortaucipir binding to CTE Type I filaments from the case with PART. In the latter, flortaucipir binds in a 1:1 molecular stoichiometry with tau, adjacent to lysine 353 and aspartate 358. By adopting a tilted geometry with respect to the helical axis, the 4.7 Å distance between neighbouring tau monomers is reconciled with the 3.5 Å distance consistent with π-π-stacking between neighbouring molecules of flortaucipir.

Vimentin single cysteine residue acts as a tunable sensor for network organization and as a key for actin remodeling in response to oxidants and electrophiles.

Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a "sensor" transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin.

Membrane-Bound Vimentin Filaments Reorganize and Elongate under Strain.

Within a cell, intermediate filaments interact with other cytoskeletal components, altogether providing the cell's mechanical stability. However, little attention has been drawn to intermediate filaments close to the plasma membrane. In this cortex configuration, the filaments are coupled and arranged in parallel to the membrane, and the question arises of how they react to the mechanical stretching of the membrane. To address this question, we set out to establish an in vitro system composed of a polydimethylsiloxane-supported lipid bilayer. With a uniaxial stretching device, the supported membrane was stretched up to 34% in the presence of a lipid reservoir that was provided by adding small unilamellar vesicles in the solution. After vimentin attachment to the membrane, we observed structural changes of the vimentin filaments in networks of different densities by fluorescence microscopy and atomic force microscopy. We found that individual filaments respond to the membrane stretching with a reorganization along the stretching direction as well as an intrinsic elongation, while in a dense network, mainly filament reorganization was observed.

Immunohistochemical analysis of the vimentin filaments in Sertoli cells is a powerful tool for the prediction of spermatogenic dysfunction.

The close interaction between male germ cells and Sertoli cells, a type of somatic cell found in the seminiferous tubules of mammalian testis, is essential for the normal progression of spermatogenesis in mammals. Vimentin is an intermediate filament protein that primarily provides mechanical support, preserves cell shape, and maintains the nuclear position, and it is often used as a marker to identify Sertoli cells. Vimentin is known to be involved in many diseases and aging processes; however, how vimentin is related to spermatogenic dysfunction and the associated functional changes is still unclear. In a previous study, we reported that vitamin E deficiency affected the testes, epididymis, and spermatozoa of mice, accelerating the progression of senescence. In this study, we focused on the Sertoli cell marker vimentin and explored the relationship between the cytoskeletal system of Sertoli cells and spermatogenic dysfunction using testis tissue sections that caused male reproductive dysfunction with vitamin E deficiency. The immunohistochemical analysis showed that the proportion of the vimentin-positive area in seminiferous tubule cross-sections was significantly increased in testis tissue sections of the vitamin E-deficient group compared with the proportion in the control group. The histological analysis of testis tissue sections from the vitamin E-deficient group showed that vimentin-positive Sertoli cells were greatly extended from the basement membrane, along with an increased abundance of vimentin. These findings suggest that vimentin may be a potential indicator for detecting spermatogenic dysfunction.

Lamin Filament Assembly Derived from the Atomic Structure of the Antiparallel Four-Helix Bundle.

The nucleoskeletal protein lamin is primarily responsible for the mechanical stability of the nucleus. The lamin assembly process requires the A11, A22, and ACN binding modes of the coiled-coil dimers. Although X-ray crystallography and chemical cross-linking analysis of lamin A/C have provided snapshots of A11 and ACN binding modes, the assembly mechanism of the entire filament remains to be explained. Here, we report a crystal structure of a coil 2 fragment, revealing the A22 interaction at the atomic resolution. The structure showed detailed structural features, indicating that two coiled-coil dimers of the coil 2 subdomain are separated and then re-organized into the antiparallel-four-helix bundle. Furthermore, our findings suggest that the ACN binding mode between coil 1a and the C-terminal part of coil 2 when the A11 tetramers are arranged by the A22 interactions. We propose a full assembly model of lamin A/C with the curvature around the linkers, reconciling the discrepancy between the in situ and in vitro observations. Our model accounts for the balanced elasticity and stiffness of the nuclear envelopes, which is essential in protecting the cellular nucleus from external pressure.

The relationship between serum neurofilament light chain and glial fibrillary acidic protein with the REM sleep behavior disorder subtype of Parkinson's disease.

Studies have shown that rapid eye movement (REM) sleep behavior disorder (RBD) is a subtype of Parkinson's disease (PD) characterized by severe cognitive impairment and rapid disease progression. However, reliable biological markers are lacking presently. Neurofilament light chain (NFL) and glial fibrillary acidic protein (GFAP) have been widely studied as biomarkers of cognition impairment. This study aimed to find biomarkers for the RBD subtype of PD by investigating the possible relationship between serum NFL, GFAP levels, and the RBD subtype. A total of 109 PD patients and 37 healthy controls (HCs) were included, and their clinical characteristics were evaluated. PD patients were divided into two groups based on whether they had probable RBD or not. Serum NFL and GFAP levels were measured using the ultrasensitive single molecule array (Simoa) platform. The obtained data were statistically analyzed using SPSS 25.0 (IBM, Chicago, IL, USA). NFL and GFAP in the PD-RBD group were elevated compared with the PD-nRBD and control groups. Moreover, serum NFL and GFAP levels positively correlated with RBD. The combination of NFL and GFAP showed good performance in identifying PD-RBD patients from PD-nRBD. After considering potential confounding factors such as age, and disease duration, serum NFL and GFAP emerged as independent risk factors for RBD. Serum NFL and GFAP were related to RBD in PD patients. Concludingly, serum NFL and GFAP might serve as promising biomarkers for the RBD subtype of PD.

Stability profile of vimentin rod domain.

Intermediate filaments (IFs) form an essential part of the metazoan cytoskeleton. Despite a long history of research, a proper understanding of their molecular architecture and assembly process is still lacking. IFs self-assemble from elongated dimers, which are defined by their central "rod" domain. This domain forms an α-helical coiled coil consisting of three segments called coil1A, coil1B, and coil2. It has been hypothesized that the structural plasticity of the dimer, including the unraveling of some coiled-coil regions, is essential for the assembly process. To systematically explore this possibility, we have studied six 50-residue fragments covering the entire rod domain of human vimentin, a model IF protein. After creating in silico models of these fragments, their evaluation using molecular dynamics was performed. Large differences were seen across the six fragments with respect to their structural variability during a 100 ns simulation. Next, the fragments were prepared recombinantly, whereby their correct dimerization was promoted by adding short N- or C-terminal capping motifs. The capped fragments were subjected to circular dichroism measurements at varying temperatures. The obtained melting temperatures reveal the relative stabilities of individual fragments, which correlate well with in silico results. We show that the least stable regions of vimentin rod are coil1A and the first third of coil2, while the structures of coil1B and the rest of coil2 are significantly more robust. These observations are in line with the data obtained using other experimental approaches, and contribute to a better understanding of the molecular mechanisms driving IF assembly.

A Ca2+-Mediated Switch of Epiplakin from a Diffuse to Keratin-Bound State Affects Keratin Dynamics.

Keratins exert important structural but also cytoprotective functions. They have to be adaptable to support cellular homeostasis. Epiplakin (EPPK1) has been shown to decorate keratin filaments in epithelial cells and to play a protective role under stress, but the mechanism is still unclear. Using live-cell imaging of epithelial cells expressing fluorescently tagged EPPK1 and keratin, we report here an unexpected dynamic behavior of EPPK1 upon stress. EPPK1 was diffusely distributed throughout the cytoplasm and not associated with keratin filaments in living cells under standard culture conditions. However, ER-, oxidative and UV-stress, as well as cell fixation, induced a rapid association of EPPK1 with keratin filaments. This re-localization of EPPK1 was reversible and dependent on the elevation of cytoplasmic Ca2+ levels. Moreover, keratin filament association of EPPK1 led to significantly reduced keratin dynamics. Thus, we propose that EPPK1 stabilizes the keratin network in stress conditions, which involve increased cytoplasmic Ca2+.

Mechanics of Single Vimentin Intermediate Filaments Under Load.

The eukaryotic cytoskeleton consists of three different types of biopolymers - microtubules, actin filaments, and intermediate filaments - and provides cells with versatile mechanical properties, combining stability and flexibility. The unique molecular structure of intermediate filaments leads to high extensibility and stability under load. With high laser power dual optical tweezers, the mechanical properties of intermediate filaments may be investigated, while monitoring the extension with fluorescence microscopy. Here, we provide detailed protocols for the preparation of single vimentin intermediate filaments and general measurement protocols for (i) stretching experiments, (ii) repeated loading and relaxation cycles, and (iii) force-clamp experiments. We describe methods for the analysis of the experimental data in combination with computational modeling approaches.

Quantifying the Interaction Strength Between Biopolymers.

The cytoskeleton consists of three types of biopolymers-actin filaments, microtubules, and intermediate filaments-and the interplay between these components is essential for many cellular functions such as cell migration, mitosis, and the mechanical response to external cues. In the cell, the interactions between the filaments are mediated by a myriad of cross-linkers and motor proteins; however, direct forces, mediated by electrostatics or hydrophobicity, may also play an important role. Here, we provide experimental protocols and approaches for analysis and modeling for studying the interactions between either two individual vimentin intermediate filaments or between a vimentin intermediate filament and a microtubule.