Dynamic structural order of a low-complexity domain facilitates assembly of intermediate filaments.

The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent on either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Drosophila Tm1-I/C IFs functions by forming labile cross-ß interactions. These interactions are seen in polymers made from the tail domain alone, as well as in assembled IFs formed by the intact Tm1-I/C protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology.

Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins.

BACKGROUND: Profilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 ("A") domain and a unique "B" domain with a nuclear localization sequence. OBJECTIVE: To determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal "AB" domain and compare it to other S100 fused-type proteins. METHODS: We used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins. RESULTS: Comparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins. CONCLUSION: The N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions.

Geigerin-induced disorganization of desmin, an intermediate filament of the cytoskeleton, in a murine myoblast cell line (C2C12).

Ingestion of large quantities of Geigeria species by sheep causes "vermeersiekte", an economically important poisoning in southern Africa. The toxic principles are several sesquiterpene lactones, such as vermeerin, geigerin and ivalin. These sesquitepene lactones are myotoxic and the disease is characterized by microscopic and ultrastructural lesions in skeletal and cardiac muscle. Murine myoblast cells (C2C12) were exposed to 2.0, 2.5 and 5.0 mM geigerin for 24, 48 and 72 h to evaluate its effect on cytoskeletal proteins and filaments using immunocytochemistry and immunofluorescence staining. A concentration-dependent cytotoxic response was observed in desmin-expressing murine myoblasts under the light microscope, evidenced by disorganization and dot-like perinuclear aggregation of desmin filaments in the cells. ß-Tubulin, other desmin-associated proteins (αB-crystallin and synemin) as well as the microfilament F-actin were unaffected. The disorganization and aggregation of desmin following exposure to increasing geigerin concentrations is significant and can explain some of the striated muscle lesions observed in "vermeersiekte".

Human alpha A-crystallin missing N-terminal domain poorly complexes with filensin and phakinin.

The aim of this study was to determine relative importance of N-terminal domain and C-terminal extension of αA-crystallin during their in vitro complex formation with phakinin and filensin (the two lens-specific intermediate filament [IF] proteins). Cloned phakinin, filensin and vimentin were purified under a denaturing conditions by consecutive DEAE-cellulose-, hydroxyapatite- and Sephadex G-75-column chromatographic methods. WTαA-crystallin, αA-NT (N-terminal domain [residue number 1-63])-deleted and αA-CT (C-terminal terminal extension [residue number 140-173]-deleted), were cloned in pET 100 TOPO vector, expressed in BL-21 (DE3) cells using 1% IPTG, and purified using a Ni2+-affinity column. The following two in vitro methods were used to determine complex formation of WT-αA, αA-NT, or αA-CT with phakinin, filensin or both phakinin plus filensin together: an ultracentrifugation sedimentation (centrifugation at 80,000 × g for 30 min at 20 °C) followed by SDS-PAGE analysis, and an electron microscopic analysis. In the first method, the individual control proteins (WT-αA, αA-NT and αA-CT crystallin species) remained in the supernatant fractions whereas phakinin, filensin, and vimentin were recovered in the pellet fractions. On complex formation by individual WT-αA-, αA-NT or αA-CT-species with filensin, phakinin or both phakinin and filensin, WT-αA and αA-CT were recovered in the pellet fraction with phakinin, filensin or both filensin and phakinin, whereas αA-NT remained mostly in the supernatant, suggesting its poor complex formation property. EM-studies showed filamentous structure formation between WT-αA and αA-CT with phakinin or filensin, or with both filensin and phakinin together but relatively poor filamentous structures with αA-NT. Together, the results suggest that the N-terminal domain of αA-crystallin is required during in vitro complex formation with filensin and phakinin.

The molecular architecture of lamins in somatic cells.

The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin. Besides providing structural stability to the nucleus, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication. However, the structural organization of the nuclear lamina is poorly understood. Here we use cryo-electron tomography to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fibre appearance and shows that A- and B-type lamins assemble into tetrameric filaments of 3.5 nm thickness. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshworks at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.

Connexin 50 Regulates Surface Ball-and-Socket Structures and Fiber Cell Organization.

PURPOSE: The roles of gap junction protein connexin 50 (Cx50) encoded by Gja8, during lens development are not fully understood. Connexin 50 knockout (KO) lenses have decreased proliferation of epithelial cells and altered fiber cell denucleation. We further investigated the mechanism for cellular defects in Cx50 KO (Gja8-/-) lenses. METHODS: Fiber cell morphology and subcellular distribution of various lens membrane/cytoskeleton proteins from wild-type and Cx50 KO mice were visualized by immunofluorescent staining and confocal microscopy. RESULTS: We observed multiple morphological defects in the cortical fibers of Cx50 KO lenses, including abnormal fiber cell packing geometry, decreased F-actin enrichment at tricellular vertices, and disrupted ball-and-socket (BS) structures on the long sides of hexagonal fibers. Moreover, only small gap junction plaques consisting of Cx46 (α3 connexin) were detected in cortical fibers and the distributions of the BS-associated beta-dystroglycan and ZO-1 proteins were altered. CONCLUSIONS: Connexin 50 gap junctions are important for BS structure maturation and cortical fiber cell organization. Connexin 50-based gap junction plaques likely form structural domains with an array of membrane/cytoskeletal proteins to stabilize BS. Loss of Cx50-mediated coupling, BS disruption, and altered F-actin in Cx50 KO fibers, thereby contribute to the small lens and mild cataract phenotypes.

Immunolocalization of scaffoldin, a trichohyalin-like protein, in the epidermis of the chicken embryo.

Recent comparative genomic studies have identified a chicken gene that codes for a trichohyalin-like protein rich in arginine and glutamic acid termed scaffoldin. Immunocytochemistry and immunoelectron microscopy show that this protein is predominantly localized in periderm granules, subcellular structures present in the periderm of the embryonic epidermis of chick scales, beak, claw, and in the sheath of developing and regenerating feathers. This suggests that scaffoldin contributes to the formation of periderm granules and to the soft cornification of the embryonic epidermis before the definitive epidermis is formed. Scaffoldin is absent from the definitive and adult epidermis generated underneath the periderm in scales and in inter-follicular regions. Scaffoldin mixes with corneous beta-proteins (beta-keratins) synthesized in keratinocytes of the transitional layers formed beneath the periderm in the subunguis of the developing claws. Immunoreactivity for scaffoldin is absent in keratinocytes that accumulate corneous beta-proteins such as those of scales, claws, and barbule-barb cells of feathers. Corneous beta-proteins represent the prevalent type of proteins present in adult epidermis of claws, scales, and feathers. These observations indicate that scaffoldin is a protein of transitional epidermal cells of the avian integument and might represent an important component of periderm granules.

Impact of ion valency on the assembly of vimentin studied by quantitative small angle X-ray scattering.

The assembly kinetics of intermediate filament (IF) proteins from tetrameric complexes to single filaments and networks depends on the protein concentration, temperature and the ionic composition of their environment. We systematically investigate how changes in the concentration of monovalent potassium and divalent magnesium ions affect the internal organization of the resulting filaments. Small angle X-ray scattering (SAXS) is very sensitive to changes in the filament cross-section such as diameter or compactness. Our measurements reveal that filaments formed in the presence of magnesium chloride differ distinctly from filaments formed in the presence of potassium chloride. The principle multi-step assembly mechanism from tetramers via unit-length filaments (ULF) to elongated filaments is not changed by the valency of ions. However, the observed differences indicate that the magnesium ions free the head domains of tetramers from unproductive interactions to allow assembly but at the same time mediate strong inter-tetrameric interactions that impede longitudinal annealing of unit-length filaments considerably, thus slowing down filament growth.

Coiling and maturation of a high-performance fibre in hagfish slime gland thread cells.

The defensive slime of hagfishes contains thousands of intermediate filament protein threads that are manufactured within specialized gland thread cells. The material properties of these threads rival those of spider dragline silks, which makes them an ideal model for biomimetic efforts to produce sustainable protein materials, yet how the thread is produced and organized within the cell is not well understood. Here we show how changes in nuclear morphology, size and position can explain the three-dimensional pattern of thread coiling in gland thread cells, and how the ultrastructure of the thread changes as very young thread cells develop into large cells with fully mature coiled threads. Our model provides an explanation for the complex process of thread assembly and organization that has fascinated and perplexed biologists for over a century, and provides valuable insights for the quest to manufacture high-performance biomimetic protein materials.

Nigrostriatal overabundance of α-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity.

α-Synuclein (α-syn) is a presynaptic protein present at most nerve terminals, but its function remains largely unknown. The familial forms of Parkinson's disease associated with multiplications of the α-syn gene locus indicate that overabundance of this protein might have a detrimental effect on dopaminergic transmission. To investigate this hypothesis, we use adeno-associated viral (AAV) vectors to overexpress human α-syn in the rat substantia nigra. Moderate overexpression of either wild-type (WT) or A30P α-syn differs in the motor phenotypes induced, with only the WT form generating hemiparkinsonian impairments. Wild-type α-syn causes a reduction of dopamine release in the striatum that exceeds the loss of dopaminergic neurons, axonal fibers, and the reduction in total dopamine. At the ultrastructural level, the reduced dopamine release corresponds to a decreased density of dopaminergic vesicles and synaptic contacts in striatal terminals. Interestingly, the membrane-binding-deficient A30P mutant does neither notably reduce dopamine release nor it cause ultrastructural changes in dopaminergic axons, showing that α-syn's membrane-binding properties are critically involved in the presynaptic defects. To further determine if the affinity of the protein for membranes determines the extent of motor defects, we compare three forms of α-syn in conditions leading to pronounced degeneration. While membrane-binding α-syns (wild-type and A53T) induce severe motor impairments, an N-terminal deleted form with attenuated affinity for membranes is inefficient in inducing motor defects. Overall, these results demonstrate that α-syn overabundance is detrimental to dopamine neurotransmission at early stages of the degeneration of nigrostriatal dopaminergic axons.

Schwann cells can be reprogrammed to multipotency by culture.

Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription-polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75(NTR), and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-ß were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions.

A novel terminal web-like structure in cortical lens fibers: architecture and functional assessment.

This study describes a novel cytoskeletal array in fiber cells of the ocular lens of the rat and shows its relationship to the classical terminal web of other epithelial tissues. Naive adult Sprague-Dawley rats (n = 28) were utilized. F-actin, fodrin, myosin IIA, and CP49 distribution was assessed in anterior and posterior polar sections. For functional analysis, lenses were cultured with or without cytochalasin-D for 3 hr, then processed for confocal microscopy or assessed by laser scan analysis along sutures. Phalloidin labeling demonstrated a dense mesh of F-actin adjacent to posterior sutural domains to a subcapsular depth of 400 µm. Anterior polar sections revealed a comparable actin structure adjacent to anterior suture branches however, it was not developed in superficial fibers. Fodrin and myosin were localized within the web-like actin apparatus. The data was used to construct a model showing that the cytoskeletal array is located within the blunt, variable-width fiber ends that abut at sutures such that the "terminal web" flanks the suture on either side. Treatment with cytochalasin-D resulted in partial disassembly of the "terminal web" and perturbed cellular organization. Laser scan analysis revealed that cytochalasin-D treated lenses had significantly greater focal variability than control lenses (P = 0.020). We conclude that cortical fibers of rat lenses contain a bipolar structure that is structurally and compositionally analogous to classical terminal webs. The results indicate that the lens "terminal web" functions to stabilize lens fiber ends at sutures thus minimizing structural disorder, which in turn, promotes the establishment and maintenance of lens transparency.

The intermediate filament network in cultured human keratinocytes is remarkably extensible and resilient.

The prevailing model of the mechanical function of intermediate filaments in cells assumes that these 10 nm diameter filaments make up networks that behave as entropic gels, with individual intermediate filaments never experiencing direct loading in tension. However, recent work has shown that single intermediate filaments and bundles are remarkably extensible and elastic in vitro, and therefore well-suited to bearing tensional loads. Here we tested the hypothesis that the intermediate filament network in keratinocytes is extensible and elastic as predicted by the available in vitro data. To do this, we monitored the morphology of fluorescently-tagged intermediate filament networks in cultured human keratinocytes as they were subjected to uniaxial cell strains as high as 133%. We found that keratinocytes not only survived these high strains, but their intermediate filament networks sustained only minor damage at cell strains as high as 100%. Electron microscopy of stretched cells suggests that intermediate filaments are straightened at high cell strains, and therefore likely to be loaded in tension. Furthermore, the buckling behavior of intermediate filament bundles in cells after stretching is consistent with the emerging view that intermediate filaments are far less stiff than the two other major cytoskeletal components F-actin and microtubules. These insights into the mechanical behavior of keratinocytes and the cytokeratin network provide important baseline information for current attempts to understand the biophysical basis of genetic diseases caused by mutations in intermediate filament genes.

Rheological and structural properties of dilute active filament solutions.

The rheology and the structure of a dilute semiflexible biofilament solution, like F-actin, interacting via molecular motors is probed by molecular dynamics simulations. Oscillatory external shear is used to measure the storage and loss moduli as a function of motor activity in a range of frequencies and for low shear rates. The overall effect of the motor activity on the rheological properties is interpreted as an increase of the temperature, with the effective temperature proportional to the density of motors. However, the effect of motors on the structural properties of the solution, such as the orientation correlation function, is opposite: the motors drastically increase the orientation correlation length whereas thermal fluctuations decrease it.

Identifying the role of specific motifs in the lens fiber cell specific intermediate filament phakosin.

PURPOSE: Phakosin and filensin are lens fiber cell-specific intermediate filament (IF) proteins. Unlike every other cytoplasmic IF protein, they assemble into a beaded filament (BF) rather than an IF. Why the lens fiber cell requires two unique IF proteins and why and how they assemble into a structure other than an IF are unknown. In this report we test specific motifs/domains in phakosin to identify changes that that have adapted phakosin to lens-specific structure and function. METHODS: Phakosin shows the highest level of sequence identity to K18, whose natural assembly partner is K8. We therefore exchanged conserved keratin motifs between phakosin and K18 to determine whether phakosin's divergent motifs could redirect the assembly of chimeric K18 and K8. Modified proteins were bacterially expressed and purified. Assembly competence was assessed by electron microscopy. RESULTS: Substitution of the phakosin helix initiation motif (HIM) into K18 does not alter assembly with K8, establishing that the radical divergence in phakosin HIM is not by itself the mechanism by which IF assembly is redirected to BF assembly. Unexpectedly, K18 bearing phakosin HIM resulted in normal IF assembly, despite the presence of an otherwise disease-causing R-C substitution, and two helix-disrupting glycines. This disproves the widely held belief that mutation of the R is catastrophic to IF assembly. Additional data are presented that suggest normal IF assembly is dependent on sequence-specific interactions between the IF head domain and the HIM. CONCLUSIONS: In the lens fiber cell, two members of the IF family have evolved to produce BFs instead of IFs, a change that presumably adapts the IF to a fiber cell-specific function. The authors establish here that the most striking divergence seen in phakosin is not, as hypothesized, the cause of this altered assembly outcome. The authors further establish that the HIM of IFs is far more tolerant of mutations, such as those that cause some corneal dystrophies and Alexander disease, than previously hypothesized and that normal assembly involves sequence-specific interactions between the head domain and the HIM.

Towards a molecular description of intermediate filament structure and assembly.

Intermediate filaments (IFs) represent one of the prominent cytoskeletal elements of metazoan cells. Their constituent proteins are coded by a multigene family, whose members are expressed in complex patterns that are controlled by developmental programs of differentiation. Hence, IF proteins found in epidermis differ significantly from those in muscle or neuronal tissues. Due to their fibrous nature, which stems from a fairly conserved central alpha-helical coiled-coil rod domain, IF proteins have long resisted crystallization and thus determination of their atomic structure. Since they represent the primary structural elements that determine the shape of the nucleus and the cell more generally, a major challenge is to arrive at a more rational understanding of how their nanomechanical properties effect the stability and plasticity of cells and tissues. Here, we review recent structural results of the coiled-coil dimer, assembly intermediates and growing filaments that have been obtained by a hybrid methods approach involving a rigorous combination of X-ray crystallography, small angle X-ray scattering, cryo-electron tomography, computational analysis and molecular modeling.

The last twenty residues in the head domain of mouse lamin A contain important structural elements for formation of head-to-tail polymers in vitro.

Nuclear lamins are a type of intermediate filament (IF) proteins. They have a characteristic tripartite domain structure with a alpha-helical rod domain flanked by non-alpha-helical N-terminal head and C-terminal tail domains. While the head domain has been shown to be important for the formation of head-to-tail polymers that are critical assembly intermediates for lamin IFs, essential structural elements in this domain have remained obscure. As a first step to remedy this, a series of mouse lamin A mutants in which the head domain (30 amino acid residues) was deleted stepwise from the N-terminus at intervals of 10 residues were bacterially expressed. The assembly properties in vitro of the purified recombinant proteins were explored by electron microscopy. We observed that while a lamin A mutant lacking N-terminal 10 residues formed head-to-tail polymers, a mutant lacking N-terminal 20 residues or the whole head domain (30 residues) showed significantly decreased potency to form head-to-tail polymers. These results suggest that the last 20 residues (from Arg-11 to Gln-30) of the head domain of mouse lamin A contain essential structures for the formation of head-to-tail polymers. The last 20 residues of the head domain include several conserved residues between A- and B-type lamins and also the phosphorylation site for cdc2 kinase, which affects lamin IF organization in vivo and in vitro. Our results provide clues to the molecular mechanism by which the head domain plays a crucial role in lamin polymerization.

Intermediate filaments: a role in epithelial polarity.

Intermediate filaments have long been considered mechanical components of the cell that provide resistance to deformation stress. Practical experimental problems, including insolubility, lack of good pharmacological antagonists, and the paucity of powerful genetic models have handicapped the research of other functions. In single-layered epithelial cells, keratin intermediate filaments are cortical, either apically polarized or apico-lateral. This review analyzes phenotypes of genetic manipulations of simple epithelial cell keratins in mice and Caenorhabditis elegans that strongly suggest a role of keratins in apico-basal polarization and membrane traffic. Published evidence that intermediate filaments can act as scaffolds for proteins involved in membrane traffic and signaling is also discussed. Such a scaffolding function would generate a highly polarized compartment within the cytoplasm of simple epithelial cells. While in most cases mechanistic explanations for the keratin-null or overexpression phenotypes are still missing, it is hoped that investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments.

Biomechanical properties of intermediate filaments: from tissues to single filaments and back.

The animal cell cytoskeleton consists of three interconnected filament systems: actin-containing microfilaments (MFs), microtubules (MTs), and the lesser known intermediate filaments (IFs). All IF proteins share a common tripartite domain structure and the ability to assemble into 8-12 nm wide filaments. Electron microscopy data suggest that IFs are built according to a completely different plan from that of MFs and MTs. IFs are known to impart mechanical stability to cells and tissues but, until recently, the biomechanical properties of single IFs were unknown. However, with the discovery of naturally occurring micrometer-wide IF bundles and the development of new methodologies to mechanically probe single filaments, it is now possible to propose a more unified view of IF biomechanics. Unlike MFs and MTs, single IFs can now be described as flexible, extensible and tough, which has important implications for our understanding of cell and tissue mechanics. Furthermore, the molecular mechanisms at play when IFs are deformed point toward a pivotal role for them in mechanotransduction.