Intermediate Filament Network Research Articles

Active superelasticity in three-dimensional epithelia of controlled shape.

Fundamental biological processes are carried out by curved epithelial sheets that enclose a pressurized lumen. How these sheets develop and withstand three-dimensional deformations has remained unclear. Here we combine measurements of epithelial tension and shape with theoretical modelling to show that epithelial sheets are active superelastic materials. We produce arrays of epithelial domes with controlled geometry. Quantification of luminal pressure and epithelial tension reveals a tensional plateau over several-fold areal strains. These extreme strains in thetissue are accommodated by highly heterogeneous strains at a cellular level, in seeming contradiction to the measured tensional uniformity. This phenomenon is reminiscent of superelasticity, a behaviour that is generally attributed to microscopic material instabilities in metal alloys. We show that in epithelial cells this instability is triggered by a stretch-induced dilution of the actin cortex, and is rescued by the intermediate filament network. Our study reveals a type of mechanical behaviour-which we term active superelasticity-that enables epithelial sheets to sustain extreme stretching under constant tension.

Intercalated discs: cellular adhesion and signaling in heart health and diseases.

Intercalated discs (ICDs) are highly orchestrated structures that connect neighboring cardiomyocytes in the heart. Three major complexes are distinguished in ICD: desmosome, adherens junction (AJ), and gap junction (GJ). Desmosomes are major cell adhesion junctions that anchor cell membrane to the intermediate filament network; AJs connect the actin cytoskeleton of adjacent cells; and gap junctions metabolically and electrically connect the cytoplasm of adjacent cardiomyocytes. All these complexes work as a single unit, the so-called area composita, interdependently rather than individually. Mutation or altered expression of ICD proteins results in various cardiac diseases, such as ARVC (arrhythmogenic right ventricular cardiomyopathy), dilated cardiomyopathy, and hypotrophy cardiomyopathy, eventually leading to heart failure. In this article, we first review the recent findings on the structural organization of ICD and their functions and then focus on the recent advances in molecular pathogenesis of the ICD-related heart diseases, which include two major areas: i) the ICD gene mutations in cardiac diseases, and ii) the involvement of ICD proteins in signal transduction pathways leading to myocardium remodeling and eventual heart failure. These major ICD-related signaling pathways include Wnt/β-catenin pathway, p38 MAPK cascade, Rho-dependent serum response factor (SRF) signaling, calcineurin/NFAT signaling, Hippo kinase cascade, etc., which are differentially regulated in pathological conditions.

The vimentin intermediate filament network restrains regulatory T cell suppression of graft-versus-host disease.

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T cells, in which protein kinase C-θ (PKC-θ) localizes to the contact point between T cells and antigen-presenting cells, in human and mouse Tregs, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Tregs with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Tregs were significantly better than controls at suppressing alloreactive T cell priming in graft-versus-host disease (GVHD) and GVHD lethality, using a complete MHC-mismatch mouse model of acute GVHD (C57BL/6 donor into BALB/c host). Interestingly, vimentin disruption augmented the suppressor function of PKC-θ-deficient mouse Tregs. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrate that vimentin is a key metabolic and functional controller of Treg activity and provide proof of principle that disruption of vimentin is a feasible, translationally relevant method to enhance Treg potency.

Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts.

Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks that participate in polarized cell protrusion, adhesion, and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarization and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, glial fibrillary acidic protein, and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organization and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarization of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localization of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration in astrocytes by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers, and by contributing to the maintenance of lateral cell-cell interactions.

Melt‐Compounded Keratin‐TPU Self‐Assembled Composite Film as Bioinspired e‐Skin

AbstractInspired by the keratin–elastin composition of human skin, here an artificial skin (alias electronic (e)‐skin) is developed through solvent‐free extrusion based melt mixing of thermoplastic polyurethane (TPU) with the biowaste human hair keratin. Self‐assembled composite film of TPU and keratin (i.e., PUK) shows leg skin equivalent coefficient of friction value of 0.26 ± 0.05, cheek skin equivalent average surface roughness (Ra) of 0.047 ± 0.07 µm, cytoskeletal keratin intermediate filament network like rheological behavior, porous morphology, and skin like positive triboelectric property, when the keratin content is 10 wt%. H‐bonded self‐assembled network and lubricating behavior of cysteine‐rich keratin provide such tribological and rheological behaviors of the PUK. Single electrode triboelectric nanogenerator (STENG) made of PUK containing 10 wt% keratin (i.e., 10% PUK) not only shows clear discrimination between the touch feeling sensations of bare, and glove protected human finger, by producing output voltage of ≈1 and ≈16 V cm−2, respectively, but it also can discriminate the tactile sensations of different objects viz., aluminum foil, cotton glove, wood, polyimide, and poly(tetrafluoroethylene) by producing different output voltages, resembling that the PUK composite film is well fitted for e‐skin applications.

Mouse Keratinocytes Without Keratin Intermediate Filaments Demonstrate Substrate Stiffness Dependent Behaviors.

Traditionally thought to serve active vs. passive mechanical functions, respectively, a growing body of evidence suggests that actin microfilament and keratin intermediate filament (IF) networks, together with their associated cell-cell and cell-matrix anchoring junctions, may have a large degree of functional interdependence. Therefore, we hypothesized that the loss of keratin IFs in a knockout mouse keratinocyte model would affect the kinematics of colony formation, i.e., the spatiotemporal process by which individual cells join to form colonies and eventually a nascent epithelial sheet. Time-lapse imaging and deformation tracking microscopy was used to observe colony formation for both wild type (WT) and keratin-deficient knockout (KO) mouse keratinocytes over 24h. Cells were cultured under high calcium conditions on collagen-coated substrates with nominal stiffnesses of ~ 1.2kPa (soft) and 24kPa (stiff). Immunofluorescent staining of actin and selected adhesion proteins was also performed. The absence of keratin IFs markedly affected cell morphology, spread area, and cytoskeleton and adhesion protein organization on both soft and stiff substrates. Strikingly, an absence of keratin IFs also significantly reduced the ability of mouse keratinocytes to mechanically deform the soft substrate. Furthermore, KO cells formed colonies more efficiently on stiff vs. soft substrates, a behavior opposite to that observed for WT keratinocytes. Collectively, these data are strongly supportive of the idea that an interdependence between actin microfilaments and keratin IFs does exist, while further suggesting that keratin IFs may represent an important and under-recognized component of keratinocyte mechanosensation and the force generation apparatus.

P-130 - The conserved cysteine residue of type III intermediate filaments serves as a structural element and redox sensor

Glial fibrillary acidic protein (GFAP) along with vimentin, desmin and peripherin constitutes the type III class of intermediate filament proteins, which are expressed in a cell type-dependent manner. GFAP and vimentin play a role in organelle positioning, cell migration and mechanical resistance and possess a conserved cysteine residue (cys328 in vimentin and cys294 in GFAP). We previously showed that cys328 of vimentin is critical for stress sensing, since mutants lacking this residue are resistant to vimentin reorganization induced by oxidants and electrophilic lipids. We have observed that GFAP cys294 is also a target for lipoxidation, which induces a drastic filament rearrangement in cells. Therefore, the conserved cysteine residues of type III intermediate filaments emerge as important players in cellular responses to oxidative damage. Interestingly, cysteine to serine mutations compromise assembly and copolymerization of GFAP and vimentin, showing that these residues are important for the structure of the intermediate filament network. This raises the hypothesis that modifications of cysteine residues of intermediate filaments may contribute to cellular dysfunction under pathophysiological conditions.

SMAD6 overexpression leads to accelerated myogenic differentiation of LMNA mutated cells

LMNA gene encodes lamins A and C, two major components of the nuclear lamina, a network of intermediate filaments underlying the inner nuclear membrane. Most of LMNA mutations are associated with cardiac and/or skeletal muscles defects. Muscle laminopathies include Emery-Dreifuss Muscular Dystrophy, Limb-Girdle Muscular Dystrophy 1B, LMNA-related Congenital Muscular Dystrophy and Dilated Cardiomyopathy with conduction defects. To identify potential alterations in signaling pathways regulating muscle differentiation in LMNA-mutated myoblasts, we used a previously described model of conditionally immortalized murine myoblasts: H-2K cell lines. Comparing gene expression profiles in wild-type and Lmna∆8–11 H-2K myoblasts, we identified two major alterations in the BMP (Bone Morphogenetic Protein) pathway: Bmp4 downregulation and Smad6 overexpression. We demonstrated that these impairments lead to Lmna∆8–11 myoblasts premature differentiation and can be rescued by downregulating Smad6 expression. Finally, we showed that BMP4 pathway defects are also present in myoblasts from human patients carrying different heterozygous LMNA mutations.

The desmin network is a determinant of the cytoplasmic stiffness of myoblasts.

The mechanical properties of cells are essential to maintain their proper functions, and mainly rely on their cytoskeleton. A lot of attention has been paid to actin filaments, demonstrating their central role in the cells mechanical properties, but much less is known about the participation of intermediate filament (IF) networks. Indeed the contribution of IFs, such as vimentin, keratins and lamins, to cell mechanics has only been assessed recently. We study here the involvement of desmin, an IF specifically expressed in muscle cells, in the rheology of immature muscle cells. Desmin can carry mutations responsible for a class of muscle pathologies named desminopathies. In this study, using three types of cell rheometers, we assess the consequences of expressing wild-type (WT) or mutated desmin on the rheological properties of single myoblasts. We find that the mechanical properties of the cell cortex are not correlated to the quantity, nor the quality of desmin expressed. On the contrary, the overall cell stiffness increases when the amount of WT or mutated desmin polymerised in cytoplasmic networks increases. However, myoblasts become softer when the desmin network is partially depleted by the formation of aggregates induced by the expression of a desmin mutant. We demonstrate that desmin plays a negligible role in the mechanical properties of the cell cortex but is a determinant of the overall cell stiffness. More particularly, desmin participates to the cytoplasm viscoelasticity. Desminopathies are associated with muscular weaknesses attributed to a disorganisation of the structure of striated muscle that impairs the active force generation. The present study evidences for the first time the key role of desmin in the rheological properties of myoblasts, raising the hypothesis that desmin mutations could also alter the passive mechanical properties of muscles, thus participating to the lack of force build up in muscle tissue.

Inflammatory Proteomic Network Analysis of Statin-treated and Lipopolysaccharide-activated Macrophages

A significant component of immune biology research is the investigation of protein encoding genes that play central roles in contributing inflammatory response. A gel-free quantitative bottom-up proteomics study was performed on immune cell macrophages after the combined treatment of lipopolysaccharide (LPS) and statin drugs using mass spectrometry and a detailed bioinformatics analyses were conducted. Systematic bioinformatics analysis was applied for discovering novel relationships among proteins and effects of statin and lipopolysaccharide in macrophage cells. Based on gene ontology, majority of protein encoding genes was involved in metabolic and cellular processes and are actively associated with binding, structural molecular, and catalytic activity. Notably, proteomic data analyzed by Ingenuity Pathway Analysis (IPA), discovered the plectin and prohibitin 2 protein interactions network and inflammatory-disease based protein networks. Two up-regulated proteins, plectin and prohibitin 2, were further validated by immunoblotting. Plectin was also cross-validated by immunocytochemistry, since its expression was highly modulated by statin but inhibited during LPS-stimulation. Collectively, the significant up-regulation of plectin due to the treatment of statin, suggests that statin has a significant impact on the cytoskeletal networks of cells. Plectin might have a significant role in the intermediate filament assembly and dynamics, and possibly stabilizing and crosslinking intermediate filament networks.

High content image analysis reveals function of miR-124 upstream of Vimentin in regulating motor neuron mitochondria

microRNAs (miRNAs) are critical for neuronal function and their dysregulation is repeatedly observed in neurodegenerative diseases. Here, we implemented high content image analysis for investigating the impact of several miRNAs in mouse primary motor neurons. This survey directed our attention to the neuron-specific miR-124, which controls axonal morphology. By performing next generation sequencing analysis and molecular studies, we characterized novel roles for miR-124 in control of mitochondria localization and function. We further demonstrated that the intermediate filament Vimentin is a key target of miR-124 in this system. Our data establishes a new pathway for control of mitochondria function in motor neurons, revealing the value of a neuron-specific miRNA gene as a mechanism for the re-shaping of otherwise ubiquitously-expressed intermediate filament network, upstream of mitochondria activity and cellular metabolism.

Regulation of the Actin Cytoskeleton Transformation in the Cell by ARP2/3 Complex. Review

The cytoskeleton is formed by a network of protein filaments, including microtubules, actin filaments and intermediate filaments. Filaments permeate the entire cytoplasm; they are involved in maintaining the cell shape, they organize and anchor the organelles, they control the transport of various molecules, cell division and provide signal transduction. To implement these diverse and complex functions, the components of the cytoskeleton must be very dynamic and mobile, be able to rebuilt quickly and interact with each other. This is due to the presence of a large number of actin-binding proteins—nucleators, activators, inactivators of polymerization and depolymerization of actin filaments. This review describes the regulation of actin dynamics by the Arp2/3 complex. In the cell, this complex is in an inactive state. Its activation occurs after it’s interaction with activators. Activators change the conformation and spatial arrangement of the domains of the Arp2/3 complex, providing its interaction with the monomeric and polymeric actin. Activators of the Arp2/3 complex have been known for a long time and include such proteins as WASp and WAVE. All activators possess a specific VCA domain, which is responsible for their binding to the Arp2/3 complex. The structure of the complex with bound activators has been studied using various physical-chemical methods. The inactivators of the complex only recently attracted specific attention of the investigators. At present, at least five different proteins are known to inactivate the Arp2/3 complex by binding to its various subunits. Examples of inactivators are coronin, Gmf and arpin. The structure of the Arp2/3 complex with inactivators was recently published and showed that despite their binding to different subunits of the complex, all inactivators transform the Arp2/3 complex into an “open” state, moving the actin-like Arp subunits apart from each other. Studies of the spatial organization of actin-binding proteins are necessary for understanding the patterns of interaction between them while providing the vital activity of the cell. These data can later be used in the search for new ligands to prevent metastasis of tumor cells.

Checkpoint kinase 1–induced phosphorylation of O-linked β-N-acetylglucosamine transferase regulates the intermediate filament network during cytokinesis

Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified O-linked β-N-acetylglucosamine (O-GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT-pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular O-GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an siOGT-based approach. Lastly, expression of plasmids resistant to siOGT efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1-OGT-vimentin pathway that regulates the intermediate filament network during cytokinesis.

A rim-and-spoke hypothesis to explain the biomechanical roles for cytoplasmic intermediate filament networks.

Textbook images of keratin intermediate filament (IF) networks in epithelial cells and the functional compromization of the epidermis by keratin mutations promulgate a mechanical role for this important cytoskeletal component. In stratified epithelia, keratin filaments form prominent radial spokes that are focused onto cell-cell contact sites, i.e. the desmosomes. In this Hypothesis, we draw attention to a subset of keratin filaments that are apposed to the plasma membrane. They form a rim of filaments interconnecting the desmosomes in a circumferential network. We hypothesize that they are part of a rim-and-spoke arrangement of IFs in epithelia. From our review of the literature, we extend this functional role for the subplasmalemmal rim of IFs to any cell, in which plasma membrane support is required, provided these filaments connect directly or indirectly to the plasma membrane. Furthermore, cytoplasmic IF networks physically link the outer nuclear and plasma membranes, but their participation in mechanotransduction processes remain largely unconsidered. Therefore, we also discuss the potential biomechanical and mechanosensory role(s) of the cytoplasmic IF network in terms of such a rim (i.e. subplasmalemmal)-and-spoke arrangement for cytoplasmic IF networks.

Increased Neuronal Differentiation of Neural Progenitor Cells Derived from Phosphovimentin-Deficient Mice

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIMSA/SA) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIMSA/SA astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIMSA/SA astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIMSA/SA neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.

P.179 - PLEC gene mutations cause familial disto-proximal myopathy and long QT syndrome mimicking mitochondrial disease

Plectin, a giant multifunctional cytolinker protein, plays a crucial role in orchestrating intermediate filament networks in a wide variety of tissues, as epithelia, skeletal muscle and heart. Mutations of the plectin gene (PLEC) on chromosome 8q24 cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), EBS-MD with myasthenic features, EBS with pyloric atresia, EBS-Ogna, and LGMD2Q. Here we report an Italian family with a distinctive muscle phenotype and autosomal recessive transmission in which Whole Exome Sequencing identified homozygous mutations in PLEC gene. Three brothers (two female and one male), age 52, 51 and 46 years, were affected in their thirties by adult-onset myopathy characterized by progressive disto-proximal symmetrical muscle weakness, more severe in distal muscles and upper limbs, mild facial weakness, ptosis and strabismus. There were no skin abnormalities. Cardiac investigation showed arrhythmia and long QT syndrome requiring in one patient an Implantable Cardioverter Defibrillator. Cardiac MRI demonstrated late-gadolinium enhancement in the basal inferolateral part of left ventricle, with sub-epicardic distribution pattern and intramural involvement. A fourth sister had died of sudden death at age 16 years. Myopathological analyses showed the typical hallmarks of mitochondrial diseases with numerous RRFs, a chronic myopathic process with necrosis and regeneration, desmin accumulation in some fibers and markedly reduced plectin staining. Muscle respiratory chain activities were normal. This report expands the phenotypes of plectin-associated disorders and supports the hypothesis that mitochondrial dysfunction contributes to the progression and severity of muscle damage in plectin myopathy. “Plectinopathies” represent a diagnostic challenge for their chameleon presentation and this study supports the value of next generation technologies to improve our abilities in diagnosing complex neuromuscular disorders. Analysis of PLEC should be included in the workout of mitochondrial disorders and distal myopathies.

Intracellular Motility of Intermediate Filaments.

SUMMARYThe establishment and continuous cell type-specific adaptation of cytoplasmic intermediate filament (IF) networks are linked to various types of IF motility. Motor protein-driven active transport, linkage to other cellular structures, diffusion of small soluble subunits, and intrinsic network elasticity all contribute to the motile behavior of IFs. These processes are subject to regulation by multiple signaling pathways. IF motility is thereby connected to and involved in many basic cellular processes guarding the maintenance of cell and tissue integrity. Disturbances of IF motility are linked to diseases that are characterized by cytoplasmic aggregates containing IF proteins together with other cellular components.

The desmoplakin-intermediate filament linkage regulates cell mechanics.

The translation of mechanical forces into biochemical signals plays a central role in guiding normal physiological processes during tissue development and homeostasis. Interfering with this process contributes to cardiovascular disease, cancer progression, and inherited disorders. The actin-based cytoskeleton and its associated adherens junctions are well-established contributors to mechanosensing and transduction machinery; however, the role of the desmosome-intermediate filament (DSM-IF) network is poorly understood in this context. Because a force balance among different cytoskeletal systems is important to maintain normal tissue function, knowing the relative contributions of these structurally integrated systems to cell mechanics is critical. Here we modulated the interaction between DSMs and IFs using mutant forms of desmoplakin, the protein bridging these structures. Using micropillar arrays and atomic force microscopy, we demonstrate that strengthening the DSM-IF interaction increases cell-substrate and cell-cell forces and cell stiffness both in cell pairs and sheets of cells. In contrast, disrupting the interaction leads to a decrease in these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is dismantled. These data suggest that the tissue-specific variability in DSM-IF network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning forces among cytoskeletal systems.

Regulation of microtubule-associated motors drives intermediate filament network polarization.

Intermediate filaments (IFs) are key players in the control of cell morphology and structure as well as in active processes such as cell polarization, migration, and mechanoresponses. However, the regulatory mechanisms controlling IF dynamics and organization in motile cells are still poorly understood. In this study, we investigate the mechanisms leading to the polarized rearrangement of the IF network along the polarity axis. Using photobleaching and photoconversion experiments in glial cells expressing vimentin, glial fibrillary acidic protein, and nestin, we show that the distribution of cytoplasmic IFs results from a continuous turnover based on the cooperation of an actin-dependent retrograde flow and anterograde and retrograde microtubule-dependent transports. During wound-induced astrocyte polarization, IF transport becomes directionally biased from the cell center toward the cell front. Such asymmetry in the transport is mainly caused by a Cdc42- and atypical PKC-dependent inhibition of dynein-dependent retrograde transport. Our results show how polarity signaling can affect the dynamic turnover of the IF network to promote the polarization of the network itself.

Interaction of small heat shock proteins with light component of neurofilaments (NFL).

The interaction of human small heat shock protein HspB1, its point mutants associated with distal hereditary motor neuropathy, and three other small heat shock proteins (HspB5, HspB6, HspB8) with the light component of neurofilaments (NFL) was analyzed by differential centrifugation, analytical ultracentrifugation, and fluorescent spectroscopy. The wild-type HspB1 decreased the quantity of NFL in pellets obtained after low- and high-speed centrifugation and increased the quantity of NFL remaining in the supernatant after high-speed centrifugation. Part of HspB1 was detected in the pellet of NFL after high-speed centrifugation, and at saturation, 1mol of HspB1 monomer was bound per 2mol of NFL. Point mutants of HspB1 associated with distal hereditary motor neuropathy (G84R, L99M, R140G, K141Q, and P182S) were almost as effective as the wild-type HspB1 in modulation of NFL assembly. At low ionic strength, HspB1 weakly interacted with NFL tetramers, and this interaction was increased upon salt-induced polymerization of NFL. HspB1 and HspB5 (αB-crystallin) decreased the rate of NFL polymerization measured by fluorescent spectroscopy. HspB6 (Hsp20) and HspB8 (Hsp22) were less effective than HspB1 (or HspB5) in modulation of NFL assembly. The data presented indicate that the small heat shock proteins affect NFL transition from tetramers to filaments, hydrodynamic properties of filaments, and their bundling and therefore probably modulate the formation of intermediate filament networks in neurons.