Neurofilament Biophysics: From Structure to Biomechanics.

Neurofilaments (NFs) are multisubunit, neuron-specific intermediate filaments consisting of a 10-nm diameter filament "core" surrounded by a layer of long intrinsically disordered protein (IDP) "tails." NFs are thought to regulate axonal caliber during development and then stabilize the mature axon, with NF subunit misregulation, mutation, and aggregation featuring prominently in multiple neurological diseases. The field's understanding of NF structure, mechanics, and function has been deeply informed by a rich variety of biochemical, cell biological, and mouse genetic studies spanning more than four decades. These studies have contributed much to our collective understanding of NF function in axonal physiology and disease. In recent years, however, there has been a resurgence of interest in NF subunit proteins in two new contexts: as potential blood- and cerebrospinal fluid-based biomarkers of neuronal damage, and as model IDPs with intriguing properties. Here, we review established principles and more recent discoveries in NF structure and function. Where possible, we place these findings in the context of biophysics of NF assembly, interaction, and contributions to axonal mechanics.

Temperature dependence of IR exciton emission spectra in Müller cell intermediate filaments.

Temperature dependences of IR exciton properties in Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina were studied. It was found that the widths of the spectral emission bands in the 2500 cm-1 and 5000 cm-1 energy ranges grow with temperature. It was found that temperature effects on the bandwidth may be described by thermal activation of the low-frequency vibrational modes of the IFs. The average activation energies for the two IR bands were estimated. Considering the dynamics of IR emission, its buildup time was independent on the sample temperature, while its decay time decreased with temperature. Thus, the emission decay rate increased exponentially with the sample temperature. The mechanisms explaining the observed temperature effects were proposed and discussed. Taking into account that MC IFs are capable of transmitting ATP hydrolysis energy within and between cells, with these properties being apparently common for all IFs, these IFs may be used by cells for physical energy transport and communications. As presently reported, temperature effects upon IR exciton spectra should not affect these proposed physiological functions to any significant extent. Therefore, the currently reported data are important for improving our understanding of the physical communication mechanisms operating within and between cells.

Effects of pulsed electric fields on exciton propagation efficiency along Müller cell intermediate filaments. Possible separation mechanism of high- and low-contrast images by the eye-brain system.

In the current study, we tested a possible mechanism of low- and high-contrast image component discrimination by the vertebrate eye-brain system. Apparently the eye-brain system has to discriminate between the low-contrast image component formed by light scattered within the retina, due to interaction of photons with cells and their parts, and the high-contrast image component transmitted by excitons via the quantum mechanism. Presently, effects of pulsed electric fields applied to Müller cell (MC) intermediate filaments (IFs) on the efficiency of exciton propagation were explored. The effects of both pulse duration and amplitude were recorded. These experimental results show that the eye-brain system may be using signal modulation to discriminate between high- and low-contrast image components, improving our understanding of high-contrast vision in vertebrates.

Co-expression of intermediate filaments glial fibrillary acidic protein and cytokeratin in pituitary adenoma.

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 < 0.01). Simultaneous co-expression of GFAP and cytokeratin in the same cells was demonstrated in 26 out of 326 pituitary adenomas and in all 13 pituitaries. In pituitary intermediate filaments were demonstrated in a larger area of the cytoplasm than in adenoma (p < 0.01), however, overlapping expression was seen in 2.6% of the total area in both, pituitary and adenoma. Congenially, cells with overlapping expression were found near vessels and in follicles. Furthermore, adenomas with cellular co-expression of GFAP and cytokeratin were associated with a lower recurrence rate (7.7%) compared to adenomas without co-expression of intermediate filaments (17.8%). 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.

Intermediate filaments as effectors of differentiation.

After the initial discovery of intermediate filament (IF)-forming proteins in 1968, a decade would elapse before they were revealed to comprise a diverse group of proteins which undergo tissue-, developmental stage-, differentiation-, and context-dependent regulation. Our appreciation for just how large (n = 70), conserved, complex, and dynamic IF genes and proteins are became even sharper upon completion of the human genome project. While there has been extraordinary progress in understanding the multimodal roles of IFs in cells and tissues, even revealing them as direct causative agents in a broad array of human genetic disorders, the link between individual IFs and cell differentiation has remained elusive. Here, we review evidence that demonstrates a role for IFs in lineage determination, cell differentiation, and tissue homeostasis. A major theme in this review is the function of IFs as sensors and transducers of mechanical forces, intersecting microenvironmental cues and fundamental processes through cellular redox balance.

Mechanical and Non-Mechanical Functions of Filamentous and Non-Filamentous Vimentin.

Intermediate filaments (IFs) formed by vimentin are less understood than their cytoskeletal partners, microtubules and F-actin, but the unique physical properties of IFs, especially their resistance to large deformations, initially suggest a mechanical function. Indeed, vimentin IFs help regulate cell mechanics and contractility, and in crowded 3D environments they protect the nucleus during cell migration. Recently, a multitude of studies, often using genetic or proteomic screenings show that vimentin has many non-mechanical functions within and outside of cells. These include signaling roles in wound healing, lipogenesis, sterol processing, and various functions related to extracellular and cell surface vimentin. Extracellular vimentin is implicated in marking circulating tumor cells, promoting neural repair, and mediating the invasion of host cells by viruses, including SARS-CoV, or bacteria such as Listeria and Streptococcus. These findings underscore the fundamental role of vimentin in not only cell mechanics but also a range of physiological functions. Also see the video abstract here https://youtu.be/YPfoddqvz-g.

LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments.

BACKGROUND: Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood. METHODS: Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes. RESULTS: Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation. CONCLUSIONS: LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

Cytoskeleton-a crucial key in host cell for coronavirus infection.

The emerging coronavirus (CoV) pandemic is threatening the public health all over the world. Cytoskeleton is an intricate network involved in controlling cell shape, cargo transport, signal transduction, and cell division. Infection biology studies have illuminated essential roles for cytoskeleton in mediating the outcome of host‒virus interactions. In this review, we discuss the dynamic interactions between actin filaments, microtubules, intermediate filaments, and CoVs. In one round of viral life cycle, CoVs surf along filopodia on the host membrane to the entry sites, utilize specific intermediate filament protein as co-receptor to enter target cells, hijack microtubules for transportation to replication and assembly sites, and promote actin filaments polymerization to provide forces for egress. During CoV infection, disruption of host cytoskeleton homeostasis and modification state is tightly connected to pathological processes, such as defective cytokinesis, demyelinating, cilia loss, and neuron necrosis. There are increasing mechanistic studies on cytoskeleton upon CoV infection, such as viral protein‒cytoskeleton interaction, changes in the expression and post-translation modification, related signaling pathways, and incorporation with other host factors. Collectively, these insights provide new concepts for fundamental virology and the control of CoV infection.

Withaferin-A Can Be Used to Modulate the Keratin Network of Intermediate Filaments in Human Epidermal Keratinocytes.

The mechanical state of cells is a critical part of their healthy functioning and it is controlled primarily by cytoskeletal networks (actin, microtubules and intermediate filaments). Drug-based strategies targeting the assembly of a given cytoskeletal network are often used to pinpoint their role in cellular function. Unlike actin and microtubules, there has been limited interest in the role of intermediate filaments, and fewer drugs have thus been identified and characterised as modulators of its assembly. Here, we evaluate whether Withaferin-A (WFA), an established disruptor of vimentin filaments, can also be used to modulate keratin filament assembly. Our results show that in keratinocytes, which are keratin-rich but vimentin-absent, Withaferin-A disrupts keratin filaments. Importantly, the dosages required are similar to those previously reported to disrupt vimentin in other cell types. Furthermore, Withaferin-A-induced keratin disassembly is accompanied by changes in cell stiffness and migration. Therefore, we propose that WFA can be repurposed as a useful drug to disrupt the keratin cytoskeleton in epithelial cells.

Cytoskeletal organization of axons in vertebrates and invertebrates.

The maintenance of axons for the lifetime of an organism requires an axonal cytoskeleton that is robust but also flexible to adapt to mechanical challenges and to support plastic changes of axon morphology. Furthermore, cytoskeletal organization has to adapt to axons of dramatically different dimensions, and to their compartment-specific requirements in the axon initial segment, in the axon shaft, at synapses or in growth cones. To understand how the cytoskeleton caters to these different demands, this review summarizes five decades of electron microscopic studies. It focuses on the organization of microtubules and neurofilaments in axon shafts in both vertebrate and invertebrate neurons, as well as the axon initial segments of vertebrate motor- and interneurons. Findings from these ultrastructural studies are being interpreted here on the basis of our contemporary molecular understanding. They strongly suggest that axon architecture in animals as diverse as arthropods and vertebrates is dependent on loosely cross-linked bundles of microtubules running all along axons, with only minor roles played by neurofilaments.

Crystal Structure of Keratin 1/10(C401A) 2B Heterodimer Demonstrates a Proclivity for the C-Terminus of Helix 2B to Form Higher Order Molecular Contacts.

We previously determined the crystal structure of the wild-type keratin 1/10 helix 2B heterodimer at 3.3 Å resolution. We proposed that the resolution of the diffraction data was limited due to the crystal packing effect from keratin 10 (K10) residue Cys401. Cys401K10 formed a disulfide-linkage with Cys401 from another K1/10 heterodimer, creating an "X-shaped" structure and a loose crystal packing arrangement. We hypothesized that mutation of Cys401K10 to alanine would eliminate the disulfide-linkage and improve crystal packing thereby increasing resolution of diffraction and enabling a more accurate side chain electron density map. Indeed, when a K10 Cys401Ala 2B mutant was paired with its native keratin 1 (K1) 2B heterodimer partner its x-ray crystal structure was determined at 2.07 Å resolution; the structure does not contain a disulfide linkage. Superposition of the K1/K10(Cys401Ala) 2B structure onto the wild-type K1/10 2B heterodimer structure had a root-mean-square-deviation of 1.88 Å; the variability in the atomic positions reflects the dynamic motion expected in this filamentous coiled-coil complex. The electrostatic, hydrophobic, and contour features of the molecular surface are similar to the lower resolution wild-type structure. We postulated that elimination of the disulfide linkage in the K1/K10(Cys401Ala) 2B structure could allow for the 2B heterodimers to bind/pack in the A22 tetramer configuration associated with mature keratin intermediate filament assembly. Analysis of the crystal packing revealed a half-staggered anti-parallel tetrameric complex of 2B heterodimers; however, their register is not consistent with models of the A22 mode of tetrameric alignment or prior biochemical cross-linking studies.

F-Actin Interactome Reveals Vimentin as a Key Regulator of Actin Organization and Cell Mechanics in Mitosis.

Most metazoan cells entering mitosis undergo characteristic rounding, which is important for accurate spindle positioning and chromosome separation. Rounding is driven by contractile tension generated by myosin motors in the sub-membranous actin cortex. Recent studies highlight that alongside myosin activity, cortical actin organization is a key regulator of cortex tension. Yet, how mitotic actin organization is controlled remains poorly understood. To address this, we characterized the F-actin interactome in spread interphase and round mitotic cells. Using super-resolution microscopy, we then screened for regulators of cortex architecture and identified the intermediate filament vimentin and the actin-vimentin linker plectin as unexpected candidates. We found that vimentin is recruited to the mitotic cortex in a plectin-dependent manner. We then showed that cortical vimentin controls actin network organization and mechanics in mitosis and is required for successful cell division in confinement. Together, our study highlights crucial interactions between cytoskeletal networks during cell division.

Promiscuous Dimerization Between the Caenorhabditis elegans IF Proteins and a Hypothesis to Explain How Multiple IFs Persist Over Evolutionary Time.

Our previous calculations of ionic interactions indicated that the Caenorhabditis elegans intermediate filament (IF) IFA proteins, in addition to IFA/IFB-1 heterodimers, may also form homodimers. In order to prove the significance of these calculations, we analysed the dimerization potential of the IFA chains in blot overlays. Unexpectedly, we found here that the dimerization of the IFA-1 protein was of both homotypic and heterotypic nature, and involved all proteins immobilized on the membrane (IFA-1, IFA-2, IFA-4, IFB-1, IFB-2, IFC-1, IFC-2, IFD-1, IFD-2 and IFP-1). A similar interaction profile, though less complex, was observed for two biotinylated proteins (IFA-2 and IFA-4). These and previous results indicate that the IFA proteins are able to form many different heteropolymeric and homopolymeric complexes in the C. elegans tissue, but that only those triggered by the IFA-specific IFB-1 protein result in mature IFs. Moreover, the calculations of the possible ionic interactions between the individual rod sequences as well as their various deletion variants indicated a special role in this process for the middle part of the C. elegans IF coil 1B segment that is deleted in all vertebrate cytoplasmic IFs. We hypothesized here, therefore, that the striking promiscuity of the C. elegans IFs originally involved a nuclear lamin which, due to a two-heptad-long rod deletion, prevented formation of a functional lamin/cIF dimer. This, in concert with an efficient dimerization and a strict tissue-specific co-expression, may allow expansion and maintenance of the multiple Caenorhabditis IFs. A possible implication for evolution of chordate IFs proteins is also discussed.

Neurofilaments form flexible bundles during neuritogenesis in culture and in mature axons in situ.

Neurofilaments (NFs) undergo cation-dependent phospho-mediated associations with each other and other cytoskeletal elements that support axonal outgrowth. Progressive NF-NF associations generate a resident, bundled population that undergoes exchange with transporting NFs. We examined the properties of bundled NFs. Bundles did not always display a fully linear profile but curved and twisted at various points along the neurite length. Bundles retracted faster than neurites and retracted bundles did not expand following extraction with Triton, indicating that they coiled passively rather than due to pressure from the cell. Bundles consisted of helically wound NFs, which may provide flexibility necessary for turning of growing axons during pathfinding. Interactions between NFs and other cytoskeletal elements may be disrupted en masse during neurite retraction or regionally during remodeling. It is suggested that bundles within long axons that cannot be fully retracted into the soma could provide maintain proximal support yet still allow more distal flexibility for remodeling and changing direction during pathfinding.

Amnion membrane organ-on-chip: an innovative approach to study cellular interactions.

The amnion membrane that lines the human intrauterine cavity is composed of amnion epithelial cells (AECs) connected to an extracellular matrix containing amnion mesenchymal cells (AMCs) through a basement membrane. Cellular interactions and transitions are mechanisms that facilitate membrane remodeling to maintain its integrity. Dysregulation of cellular remodeling, primarily mediated by oxidative stress (OS), is often associated with preterm birth. However, the mechanisms that maintain membrane homeostasis remain unclear. To understand these mechanisms, we developed an amnion membrane organ-on-chip (AM-OOC) and tested the interactive and transition properties of primary human AECs and AMCs under normal and OS conditions. AM-OOC contained 2 chambers connected by type IV collagen-coated microchannels, allowing independent culture conditions that permitted cellular migration and interactions. Cells grown either independently or coculture were exposed to OS inducing cigarette smoke extract, antioxidant N-acetyl-l-cysteine (NAC), or both. When grown independently, AECs transitioned to AMCs and migrated, whereas AMCs migrated without transition. OS caused AECs' transition but prevented migration, whereas AMCs' migration was unhindered. Coculture of cells facilitated transition, migration, and eventual integration in the contiguous population. OS cotreatment in both chambers facilitated AECs' transition, prevented migration, and increased inflammation, a process that was prevented by NAC. AM-OOC recapitulated cellular mechanisms observed in utero and enabled experimental manipulation of cells to determine their roles during pregnancy and parturition.-Richardson, L., Jeong, S., Kim, S., Han, A., Menon, R. Amnion membrane organ-on-chip: an innovative approach to study cellular interactions.

Analysis of neurofilament concentration in healthy adult horses and utility in the diagnosis of equine protozoal myeloencephalitis and equine motor neuron disease.

Neurofilaments (NFs) are structural proteins of neurons that are released in significant quantities in the cerebrospinal fluid and blood as a result of neuronal degeneration or axonal damage. Therefore, NFs have potential as biomarkers for neurologic disorders. Neural degeneration increases with age and has the potential to confound the utility of NFs as biomarkers in the diagnosis of neurologic disorders. We investigated this relationship in horses with and without neurological diagnosis. While controlling for horse type (draft, pleasure, and racing), we evaluated the relationship between serum heavy-chain phosphorylated neurofilaments (pNF-H) and age, sex, and serum vitamin E concentrations. Serum pNF-H concentrations increased by 0.002 ng/ml for each year increase in age. There were significant differences in the serum pNF-H concentration among the type of activity performed by the horse. The highest serum pNF-H concentration was found in horses performing heavy work activity (racehorse) and with lower serum pNF-H concentration found among light (pleasure riding) and moderate (draft) activity. There was no significant association between the pNF-H concentration and sex or vitamin E concentration. Serum pNF-H concentration was elevated among horses afflicted with EMND and EPM when compared with control horses without evidence of neurologic disorders. Accordingly, serum pNF-H concentration can serve as a useful biomarker to complement the existing diagnostic work-up of horses suspected of having EPM or EMND.

Quantitative Analysis of Nuclear Lamins Imaged by Super-Resolution Light Microscopy.

The nuclear lamina consists of a dense fibrous meshwork of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph-based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information.

The Cytoskeleton-A Complex Interacting Meshwork.

The cytoskeleton of animal cells is one of the most complicated and functionally versatile structures, involved in processes such as endocytosis, cell division, intra-cellular transport, motility, force transmission, reaction to external forces, adhesion and preservation, and adaptation of cell shape. These functions are mediated by three classical cytoskeletal filament types, as follows: Actin, microtubules, and intermediate filaments. The named filaments form a network that is highly structured and dynamic, responding to external and internal cues with a quick reorganization that is orchestrated on the time scale of minutes and has to be tightly regulated. Especially in brain tumors, the cytoskeleton plays an important role in spreading and migration of tumor cells. As the cytoskeletal organization and regulation is complex and many-faceted, this review aims to summarize the findings about cytoskeletal filament types, including substructures formed by them, such as lamellipodia, stress fibers, and interactions between intermediate filaments, microtubules and actin. Additionally, crucial regulatory aspects of the cytoskeletal filaments and the formed substructures are discussed and integrated into the concepts of cell motility. Even though little is known about the impact of cytoskeletal alterations on the progress of glioma, a final point discussed will be the impact of established cytoskeletal alterations in the cellular behavior and invasion of glioma.

Elastocapillary self-assembled neurotassels for stable neural activity recordings.

Implantable neural probes that are mechanically compliant with brain tissue offer important opportunities for stable neural interfaces in both basic neuroscience and clinical applications. Here, we developed a Neurotassel consisting of an array of flexible and high-aspect ratio microelectrode filaments. A Neurotassel can spontaneously assemble into a thin and implantable fiber through elastocapillary interactions when withdrawn from a molten, tissue-dissolvable polymer. Chronically implanted Neurotassels elicited minimal neuronal cell loss in the brain and enabled stable activity recordings of the same population of neurons in mice learning to perform a task. Moreover, Neurotassels can be readily scaled up to 1024 microelectrode filaments, each with a neurite-scale cross-sectional footprint of 3 × 1.5 µm2, to form implantable fibers with a total diameter of ~100 µm. With their ultrasmall sizes, high flexibility, and scalability, Neurotassels offer a new approach for stable neural activity recording and neuroprosthetics.