Filament-filament Interactions Research Articles

Multiscale mechanics and temporal evolution of vimentin intermediate filament networks

The cytoskeleton, an intricate network of protein filaments, motor proteins, and cross-linkers, largely determines the mechanical properties of cells. Among the three filamentous components, F-actin, microtubules, and intermediate filaments (IFs), the IF network is by far the most extensible and resilient to stress. We present a multiscale approach to disentangle the three main contributions to vimentin IF network mechanics-single-filament mechanics, filament length, and interactions between filaments-including their temporal evolution. Combining particle tracking, quadruple optical trapping, and computational modeling, we derive quantitative information on the strength and kinetics of filament interactions. Specifically, we find that hydrophobic contributions to network mechanics enter mostly via filament-elongation kinetics, whereas electrostatics have a direct influence on filament-filament interactions.

Efficient implementation of elastohydrodynamics via integral operators

The dynamics of geometrically non-linear flexible filaments play an important role in a host of biological processes, from flagella-driven cell transport to the polymeric structure of complex fluids. Such problems have historically been computationally expensive due to numerical stiffness associated with the inextensibility constraint, as well as the often non-trivial boundary conditions on the governing high-order PDEs. Formulating the problem for the evolving shape of a filament via an integral equation in the tangent angle has recently been found to greatly alleviate this numerical stiffness. The contribution of the present manuscript is to enable the simulation of non-local interactions of multiple filaments in a computationally efficient manner using the method of regularized stokeslets within this framework. The proposed method is benchmarked against a non-local bead and link model, and recent code utilizing a local drag velocity law. Systems of multiple filaments (1) in a background fluid flow, (2) under a constant body force, and (3) undergoing active self-motility are modeled efficiently. Buckling instabilities are analyzed by examining the evolving filament curvature, as well as by coarse-graining the body frame tangent angles using a Chebyshev approximation for various choices of the relevant non-dimensional parameters. From these experiments, insight is gained into how filament-filament interactions can promote buckling, and further reveal the complex fluid dynamics resulting from arrays of these interacting fibers. By examining active moment-driven filaments, we investigate the speed of worm- and sperm-like swimmers for different governing parameters. The MATLAB(R) implementation is made available as an open-source library, enabling flexible extension for alternate discretizations and different surrounding flows.

Contractility in an extensile system.

Essentially all biology is active and dynamic. Biological entities autonomously sense, compute, and respond using energy-coupled ratchets that can produce force and do work. The cytoskeleton, along with its associated proteins and motors, is a canonical example of biological active matter, which is responsible for cargo transport, cell motility, division, and morphology. Prior work on cytoskeletal active matter systems showed either extensile or contractile dynamics. Here, we demonstrate a cytoskeletal system that can control the direction of the network dynamics to be either extensile, contractile, or static depending on the concentration of filaments or weak, transient crosslinkers through systematic variation of the crosslinker or microtubule concentrations. Based on these new observations and our previously published results, we created a simple one-dimensional model of the interaction of filaments within a bundle. Despite its simplicity, our model recapitulates the observed activities of our experimental system, implying that the dynamics of our finite networks of bundles are driven by the local filament-filament interactions within the bundle. Finally, we show that contractile phases can result in autonomously motile networks that resemble cells. Our results reveal a fundamentally important aspect of cellular self-organization: weak, transient interacting species can tune their interaction strength directly by tuning the local concentration to act like a rheostat. In this case, when the weak, transient proteins crosslink microtubules, they can tune the dynamics of the network to change from extensile to contractile to static. Our experiments and model allow us to gain a deeper understanding of cytoskeletal dynamics and provide an new understanding of the importance of weak, transient interactions to soft and biological systems.

Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments.

Intermediate filament (IF) scaffolds facilitate small heat shock protein (sHSP) function, while IF function is sHSP dependent. sHSPs interact with IFs and the importance of this interaction is to maintain the individuality of the IFs and to modulate interfilament interactions both in networks and in assembly intermediates. Mutations in both sHSPs and their interacting IF proteins phenocopy each other in the human diseases they cause. This establishes a key functional relationship between these two very distinct protein families, and it also evidences the role of this cytoskeleton-chaperone complex in the cellular stress response. In this chapter, we describe the detailed experimental protocols for the preparation of purified IF proteins and sHSPs to facilitate the study in vitro of their functional interactions. In addition, we describe the detailed biochemical procedures to assess the effect of sHSP on the assembly of IFs, the binding to IFs, and the prevention of noncovalent filament-filament interactions using in vitro cosedimentation, electron microscopy, and viscosity assays. These assays are valuable research tools to study and manipulate sHSP-IF complexes in vitro and therefore to determine the structure-function detail of this complex, and how it contributes to cellular, tissue, and organismal homeostasis and the in vivo stress response.

The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament-filament interactions.

MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four α-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with λ repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament-filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition.

Multiple Sites in αB-Crystallin Modulate Its Interactions with Desmin Filaments Assembled In Vitro

The β3- and β8-strands and C-terminal residues 155–165 of αB-crystallin were identified by pin arrays as interaction sites for various client proteins including the intermediate filament protein desmin. Here we present data using 5 well-characterised αB-crystallin protein constructs with substituted β3- and β8-strands and with the C-terminal residues 155–165 deleted to demonstrate the importance of these sequences to the interaction of αB-crystallin with desmin filaments. We used electron microscopy of negatively stained samples to visualize increased interactions followed by sedimentation assays to quantify our observations. A low-speed sedimentation assay measured the ability of αB-crystallin to prevent the self-association of desmin filaments. A high-speed sedimentation assay measured αB-crystallin cosedimentation with desmin filaments. Swapping the β8-strand of αB-crystallin or deleting residues 155–165 increased the cosedimentation of αB-crystallin with desmin filaments, but this coincided with increased filament-filament interactions. In contrast, substitution of the β3-strand with the equivalent αA-crystallin sequences improved the ability of αB-crystallin to prevent desmin filament-filament interactions with no significant change in its cosedimentation properties. These data suggest that all three sequences (β3-strand, β8-strand and C-terminal residues 155–165) contribute to the interaction of αB-crystallin with desmin filaments. The data also suggest that the cosedimentation of αB-crystallin with desmin filaments does not necessarily correlate with preventing desmin filament-filament interactions. This important observation is relevant not only to the formation of the protein aggregates that contain both desmin and αB-crystallin and typify desmin related myopathies, but also to the interaction of αB-crystallin with other filamentous protein polymers.

Modeling the Self-Organization Property of Keratin Intermediate Filaments

Keratins, a group of eukaryotic intermediate filaments, have an important function toward structural support in epithelial cells and tissues. Formation of crosslinked networks, e.g., bundles, is necessary to achieve the mechanical properties necessary to sustain this vital role. In progenitor basal keratinocytes of the epidermis, intermediate filaments made of keratin 5 (K5) and keratin 14 (K14) exhibit a bundled organization. Recent findings from our laboratory showed that bundle formation can be self-mediated through interactions involving K14's non-helical tail domain and two distinct regions within the central rod domain of K5. Here, we exploit theoretical principles and computational modeling to investigate how these biochemical interactions best promote filament bundling. We develop a simple model where keratin filaments are treated as rigid rods to apply Brownian dynamics simulation. Our result shows that there should be long-range filament interactions, along with tail domain-mediated stabilization of filament-filament interactions, for stable bundle formation. Our model can also explain the difference of the mechanical properties of keratin networks between wild type and disease-causing mutant keratin proteins. The possible source of long-range filament interactions is discussed.

Nucleation geometry governs ordered actin networks structures

Actin filaments constitute one of the main components of cell cytoskeleton. Assembled into bundles in filopodia or in stress fibres, they play a pivotal role in eukaryotes during cell morphogenesis, adhesion and motility. The bundle emergence has been extensively related to specific actin regulators in vivo. Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices. However, the question of how geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures remains poorly studied. Here we demonstrate that the nucleation geometry in itself can be the principal determinant of actin-network architecture. We developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays. Shape, orientation and distance between nucleation regions control filament orientation and length, filament-filament interactions and filopodium-like bundle formation. Modelling of filament growth and interactions demonstrates that basic mechanical and probabilistic laws govern actin assembly in higher-order structures.

Oligomers of Mutant Glial Fibrillary Acidic Protein (GFAP) Inhibit the Proteasome System in Alexander Disease Astrocytes, and the Small Heat Shock Protein αB-Crystallin Reverses the Inhibition

The accumulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), in astrocytes of Alexander disease (AxD) impairs proteasome function in astrocytes. We have explored the molecular mechanism that underlies the proteasome inhibition. We find that both assembled and unassembled wild type (wt) and R239C mutant GFAP protein interacts with the 20 S proteasome complex and that the R239C AxD mutation does not interfere with this interaction. However, the R239C GFAP accumulates to higher levels and forms more protein aggregates than wt protein. These aggregates bind components of the ubiquitin-proteasome system and, thus, may deplete the cytosolic stores of these proteins. We also find that the R239C GFAP has a greater inhibitory effect on proteasome system than wt GFAP. Using a ubiquitin-independent degradation assay in vitro, we observed that the proteasome cannot efficiently degrade unassembled R239C GFAP, and the interaction of R239C GFAP with proteasomes actually inhibits proteasomal protease activity. The small heat shock protein, alphaB-crystallin, which accumulates massively in AxD astrocytes, reverses the inhibitory effects of R239C GFAP on proteasome activity and promotes degradation of the mutant GFAP, apparently by shifting the size of the mutant protein from larger oligomers to smaller oligomers and monomers. These observations suggest that oligomeric forms of GFAP are particularly effective at inhibiting proteasome activity.

Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176

The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.

Conspicuous involvement of desmin tail mutations in diverse cardiac and skeletal myopathies

Myofibrillar myopathy (MFM) encompasses a genetically heterogeneous group of human diseases caused by mutations in genes coding for structural proteins of muscle. Mutations in the intermediate filament (IF) protein desmin (DES), a major cytoskeletal component of myocytes, lead to severe forms of "desminopathy," which affects cardiac, skeletal, and smooth muscle. Most mutations described reside in the central alpha-helical rod domain of desmin. Here we report three novel mutations--c.1325C>T (p.T442I), c.1360C>T (p.R454W), and c.1379G>T (p.S460I)--located in desmin's non-alpha-helical carboxy-terminal "tail" domain. We have investigated the impact of these and four--c.1237G>A (p.E413K), c.1346A>C (p.K449T), c.1353C>G (p.I451M), and c.1405G>A (p.V469M)--previously described "tail" mutations on in vitro filament formation and on the generation of ordered cytoskeletal arrays in transfected myoblasts. Although all but two mutants (p.E413K, p.R454W) assembled into IFs in vitro and all except p.E413K were incorporated into IF arrays in transfected C2C12 cells, filament properties differed significantly from wild-type desmin as revealed by viscometric assembly assays. Most notably, when coassembled with wild-type desmin, these mutants revealed a severe disturbance of filament-formation competence and filament-filament interactions, indicating an inherent incompatibility of mutant and wild-type protein to form mixed filaments. The various clinical phenotypes observed may reflect altered interactions of desmin's tail domain with different components of the myoblast cytoskeleton leading to diminished biomechanical properties and/or altered metabolism of the individual myocyte. Our in vitro assembly regimen proved to be a very sensible tool to detect if a particular desmin mutation is able to cause filament abnormalities.

Characteristics of multiple filaments generated by femtosecond laser pulses in air: Prefocused versus free propagation

The characteristics of the multiple filaments formed by prefocused and freely propagating femtosecond laser pulses are investigated and compared. It is shown in our experiments that the diameter, length, stability, and interaction for the two cases can be quite different. The filaments formed by prefocused beam indicate dynamic spatial evolution with higher laser intensity and electron density. They have a typical diameter of 100 microm are of shorter length. In the free propagation case, the filaments exhibit interesting properties such as hundred-meter propagation distance and mm-size diameter. Moreover, only the interaction of the filaments with the energy background affects the evolution of the filaments. Filament-filament interactions such as the filament splitting and merging were not observed in this case.

The Alexander Disease–Causing Glial Fibrillary Acidic Protein Mutant, R416W, Accumulates into Rosenthal Fibers by a Pathway That Involves Filament Aggregation and the Association of αB-Crystallin and HSP27

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.

Mutation S233L in the 1B Domain of Keratin 1 Causes Epidermolytic Palmoplantar Keratoderma with “Tonotubular” Keratin

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by epidermolytic hyperkeratosis restricted to the palm and sole epidermis. The disorder is normally associated with dominant-negative mutations in the keratin 9 (K9) gene; however, a small number of cases have been reported where causative mutations were identified in the K1 gene. Here, we present two unrelated Dutch EPPK families with striking ultrastructural findings: tubular keratin structures in the cytoplasm of suprabasal cells. Similar structures were reported previously in a German EPPK family and were termed "tonotubular" keratin. After excluding the involvement of the K9 gene by complete sequencing, we identified a novel mutation, S233L, at the beginning of the 1B domain of K1 in both families. Protein expression studies in cultured cells indicated pathogenicity of this mutation. This is the first report of a genetic defect in this domain of K1. The unusual gain-of-function mutation points to a subtle role of the 1B domain in mediating filament-filament interactions with regular periodicity.

Interaction dynamics of a pair of vortex filament rings.

Vortex filament-filament interactions are believed to underlie lethal arrhythmias in cardiac tissue but their dynamics remain poorly understood. We numerically replicate an experimentally postulated reentrant filament configuration as a pair of adjacent circular filaments (scroll rings) with common symmetry axes and varying initial radii and separation distances. The interaction properties are quantified in terms of the scroll-ring lifetime T(L) and direction of initial velocity V0. Two cases were examined, differing only in the direction of the wave around the filament, and observed drastic differences in T(L) between the cases as the separation distance between the rings was decreased. We conclude that ring interactions present unexpected behaviors associated with competing interaction and decay mechanisms.

The Cardiomyopathy and Lens Cataract Mutation in αB-crystallin Alters Its Protein Structure, Chaperone Activity, and Interaction with Intermediate Filaments in Vitro

Desmin-related myopathy and cataract are both caused by the R120G mutation in alphaB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with alphaB-crystallin, and it identifies intermediate filaments as important physiological substrates for alphaB-crystallin. Using recombinant human alphaB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of alphaB-crystallin are reported. The secondary, tertiary, and quaternary structural features of alphaB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G alphaB-crystallin is also less stable than wild type alphaB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in the in vitro chaperone activity of the mutant alphaB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of alphaB-crystallin with intermediate filaments. It abolished the ability of alphaB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing alphaB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by alphaB-crystallin binding. These studies provide important insight into the mechanism of alphaB-crystallin-induced aggregation of intermediate filaments that causes disease.

Effect of filamin and controlled linear shear on the microheterogeneity of F‐Actin/Gelsolin gels

We have previously established [Cortese and Frieden, J. Cell Biol. 107:1477-1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross-linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F-actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F-actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament-filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04-1.39 s-1), and becomes very irregular at higher shear rates (greater than 10 s-1). We suggest here that actin-binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.

Structure of helical RecA-DNA complexes: Complexes formed in the presence of ATP-gamma-S or ATP

Electron micrographs of RecA-DNA filaments, formed under several different conditions, have been analyzed and the filament images reconstructed in three dimensions. In the presence of ATP and a non-hydrolyzable ATP analog, ATP-gamma-S, the RecA protein forms with DNA a right-handed helical complex with a pitch of approximately 95 Å. The most detailed view of the filament was obtained from analysis of RecA filaments on double-stranded DNA in the presence of ATP-gamma-S. There are approximately six subunits of RecA per turn of the helix, but both this number and the pitch are variable. From the examination of single filaments and filament-filament interactions, a picture of an extremely flexible protein structure emerges. The subunits of RecA protein are seen to be arranged in such a manner that the bound DNA must be partially exposed and able to come into contact with external DNA molecules. The RecA structure determined in the presence of ATP-gamma-S appears to be the same as the “pre-synaptic” state that occurs with ATP, in which there is recognition and pairing between homologous DNA molecules.

Action of cytochalasin D on cytoskeletal networks.

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end-to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.

Mechanism of action of cytochalasin B on actin

Substoichiometric cytochalasin B (CB) inhibits both the rate of actin polymerization and the interaction of actin filaments in solution. The polymerization rate is reduced by inhibition of actin monomer addition to the “barbed” end of the filaments where monomers normally add more rapidly. 2 μM CB reduces the polymerization rate by up to 90%, but has little effect on the rate of monomer addition at the slow (“pointed”) end of the filaments and no effect on the rate of filament annealing. Under most ionic conditions tested, 2 μM CB reduces the steady state high shear viscosity by 10–20% and increases the steady state monomer concentration by a factor of 2.5 or less. In addition to the effects on the polymerization process, 2 μM CB strongly reduces the low shear viscosity of actin filaments alone and actin filaments cross-linked by a variety of macromolecules. This may be due to inhibition of actin filament-filament interactions which normally contribute to network formation. Since the inhibition of monomer addition and of actin filament network formation have approximately the same CB concentration dependence, a common CB binding site, probably the barbed end of the filament, may be responsible for both effects.