Cingulin and paracingulin tether myosins-2 to junctions to mechanoregulate the plasma membrane.

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.

Potential implications of the glycosylation patterns in collagen α1(I) and α2(I) chains for fibril assembly and growth.

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.

50 Years of the steric-blocking mechanism in vertebrate skeletal muscle: a retrospective.

Fifty years have now passed since Parry and Squire proposed a detailed structural model that explained how tropomyosin, mediated by troponin, played a steric-blocking role in the regulation of vertebrate skeletal muscle. In this Special Issue dedicated to the memory of John Squire it is an opportune time to look back on this research and to appreciate John's key contributions. A review is also presented of a selection of the developments and insights into muscle regulation that have occurred in the years since this proposal was formulated.

An experimental model of contusion injury in humans.

INTRODUCTION: Contusion injuries are common in sport, but our knowledge of the responses to injury primarily come from animal studies and research using eccentric exercise. Therefore, the aim of this study was to develop a model of contusion injury in human participants and, additionally, investigate and compare physiological responses to four impact loads. METHODS: Thirty-two males were exposed to a single impact of either 4.2, 5.2, 6.2 or 7.2kg, dropped from 67 cm, on to the vastus lateralis of one leg. Maximum voluntary and electrically induced quadriceps force, and pressure pain threshold were measured, and blood sampling carried out, prior to and 30min, 24, 48 and 72h post-impact. Magnetic resonance imaging was carried out 24h post-impact to quantify oedema. RESULTS: Despite impact force with 7.2kg (1681.4 ± 235.6 N) not being different to 6.2kg (1690.7 ± 117.6 N), 7.2kg resulted in greater volume of oedema, voluntary force loss, pain and elevations in creatine kinase than the other loads. Although electrically induced force changed over time, post-hoc analysis failed to identify any changes. Interleukin-6 and prostaglandin-E2 did not change over time for any of the loads. Significant correlations were found between oedema volume, pressure pain threshold and maximum voluntary contraction force. CONCLUSIONS: This is the first experimental study to investigate traumatic loading of skeletal muscle and the subsequent physiological responses associated with contusion injuries in humans. The absence of immediate elevations in creatine kinase and changes in electrically induced force suggest impact, with forces similar to those experienced in contact sport, does not cause significant, direct damage to skeletal muscle. However, the relationship between oedema volume, changes in pressure pain threshold and maximum voluntary contraction force suggests central inhibition plays a role in contusion-related muscle dysfunction.

Keratin intermediate filament chains in the European common wall lizard (Podarcis muralis) and a potential keratin filament crosslinker.

On the basis of sequence homology with mammalian α-keratins, and on the criteria that the coiled-coil segments and central linker in the rod domain of these molecules must have conserved lengths if they are to assemble into viable intermediate filaments, a total of 28 Type I and Type II keratin intermediate filament chains (KIF) have been identified from the genome of the European common wall lizard (Podarcis muralis). Using the same criteria this number may be compared to 33 found here in the green anole lizard (Anole carolinensis) and 25 in the tuatara (Sphenodon punctatus). The Type I and Type II KIF genes in the wall lizard fall in clusters on chromosomes 13 and 2 respectively. Although some differences occur in the terminal domains in the KIF chains of the two lizards and tuatara, the similarities between key indicator residues - cysteine, glycine and proline - are significant. The terminal domains of the KIF chains in the wall lizard also contain sequence repeats commonly based on glycine and large apolar residues and would permit the fine tuning of physical properties when incorporated within the intermediate filaments. The H1 domain in the Type II chain is conserved across the lizards, tuatara and mammals, and has been related to its role in assembly at the 2-4 molecule level. A KIF-like chain (K80) with an extensive tail domain comprised of multiple tandem repeats has been identified as having a potential filament-crosslinking role.

Structures of the ß-Keratin Filaments and Keratin Intermediate Filaments in the Epidermal Appendages of Birds and Reptiles (Sauropsids).

The epidermal appendages of birds and reptiles (the sauropsids) include claws, scales, and feathers. Each has specialized physical properties that facilitate movement, thermal insulation, defence mechanisms, and/or the catching of prey. The mechanical attributes of each of these appendages originate from its fibril-matrix texture, where the two filamentous structures present, i.e., the corneous ß-proteins (CBP or ß-keratins) that form 3.4 nm diameter filaments and the α-fibrous molecules that form the 7-10 nm diameter keratin intermediate filaments (KIF), provide much of the required tensile properties. The matrix, which is composed of the terminal domains of the KIF molecules and the proteins of the epidermal differentiation complex (EDC) (and which include the terminal domains of the CBP), provides the appendages, with their ability to resist compression and torsion. Only by knowing the detailed structures of the individual components and the manner in which they interact with one another will a full understanding be gained of the physical properties of the tissues as a whole. Towards that end, newly-derived aspects of the detailed conformations of the two filamentous structures will be discussed and then placed in the context of former knowledge.

Keratin intermediate filament chains in tuatara (Sphenodon punctatus): A comparison of tuatara and human sequences.

Determination of the sequences of the keratin intermediate filament chains in tuatara has shown that these are closely akin to the α-keratins in human and other vertebrates, especially in the central, coiled-coil rod region. The domain lengths within the rod are preserved exactly, both Type I and Type II chains have been recognised, and sequence identity/homology exists between their respective chains. Nonetheless, there are characteristic differences in amino acid composition and sequence between their respective head (N-terminal) domains and their tail (C-terminal) domains, though some similarities are retained. Further, there is evidence of tandem repeats of a variety of lengths in the tuatara heads and tails indicative of sequence duplication events. These are not evident in human α-keratins and would therefore have implications for the physical attributes of the tissues in the two species. Multiple families of keratin-associated proteins that are ultra-high cysteine-rich or glycine + tyrosine-rich in human and other species do not have direct equivalents in the tuatara. Although high-sulphur proteins are present in tuatara the cysteine residue contents are significantly lower than in human. Further, no sequence homologies between the HS proteins in the two species have been found, thereby casting considerable doubt as to whether any matrix-forming high-sulphur proteins exist in tuatara. These observations may be correlated with the numerous cysteine-rich ß-keratins (corneous ß-proteins) that are present in tuatara, but which are conspicuously absent in mammals.

Structure and topology of the linkers in the conserved lepidosaur ß-keratin chain with four 34-residue repeats support an interfilament role for the central linker.

The ß-keratin chain with four 34-residue repeats that is conserved across the lepidosaurs (lizards, snakes and tuatara) contains three linker regions as well as a short, conserved N-terminal domain and a longer, more variable C-terminal domain. Earlier modelling had shown that only six classes of structure involving the four 34-residue repeats were possible. In three of these the 34-residue repeats were confined to a single filament (Classes 1, 2 and 3) whereas in the remaining three classes the repeats lay in two, three or four filaments, with some of the linkers forming interfilament connections (Classes 4, 5 and 6). In this work the members of each class of structure (a total of 20 arrangements) have been described and a comparison has been made of the topologies of each of the linker regions. This provides new constraints on the structure of the chain as a whole. Also, analysis of the sequences of the three linker regions has revealed that the central linker (and only the central linker) contains four short regions displaying a distinctive dipeptide repeat of the form (S-X)2,3 separated by short regions containing proline and cysteine residues. By analogy with silk fibroin proteins this has the capability of forming a ß-sheet-like conformation. Using the topology and sequence data the evidence suggests that the four 34-residue repeat chain adopts a Class 4a structure with a ß-sandwich in filament 1 connected through the central linker to a ß-sandwich in filament 2.

Lepidosaur ß-keratin chains with four 34-residue repeats: Modelling reveals a potential filament-crosslinking role.

ß-keratin chains contain a characteristic and homologous 34-residue sequence, which is believed to adopt a twisted ß-sheet conformation that assembles in an antiparallel manner with a similar sheet in a second chain to form a ß-sandwich. These sandwiches are, in turn, related to one another by a left-handed four-fold screw axis to generate a helical structure that forms the core of the 3.4 nm diameter filaments observed by electron microscopy and deduced from X-ray fibre diffraction. Recently, it has been shown that one ß-keratin chain, with a molecular weight approximately twice that of the majority of ß-keratin chains, is conserved across the lepidosaurs (lizards, snakes and tuatara). Uniquely, it contains four 34-residue repeats. Although this chain is a minor component the observation that the entire chain shows a high degree of sequence conservation between species suggests an important structural/functional role in vivo. Modelling shows that only six families of structures are physically possible. In three of these the repeats exist within a single filament and might therefore act in a filament nucleation role. In the second three families the repeats exist in two, three or four filaments, implying that their function may be to act as an inter-filament crosslinker, thereby providing lateral reinforcement to the epidermal appendage. The favoured model is one in which the first two repeats form a ß-sandwich in one filament and the second two repeats form a ß-sandwich in a neighbouring filament. Links between alternating up- and down-pointing ß-sheets would provide optimum connectivity.

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.

Molecular structure of sauropsid ß-keratins from tuatara (Sphenodon punctatus).

The birds and reptiles, collectively known as the sauropsids, can be subdivided phylogenetically into the archosaurs (birds, crocodiles), the testudines (turtles), the squamates (lizards, snakes) and the rhynchocephalia (tuatara). The structural framework of the epidermal appendages from the sauropsids, which include feathers, claws and scales, has previously been characterised by electron microscopy, infrared spectroscopy and X-ray diffraction analyses, as well as by studies of the amino acid sequences of the constituent ß-keratin proteins (also referred to as the corneous ß-proteins). An important omission in this work, however, was the lack of sequence and structural data relating to the epidermal appendages of the rhynchocephalia (tuatara), one of the two branches of the lepidosaurs. Considerable effort has gone into sequencing the tuatara genome and while this is not yet complete, there are now sufficient sequence data for conclusions to be drawn on the similarity of the ß-keratins from the tuatara to those of other members of the sauropsids. These results, together with a comparison of the X-ray diffraction pattern of tuatara claw with those from seagull feather and goanna claw, confirm that there is a common structural plan in the ß-keratins of all of the sauropsids, and not just those that comprise the archosaurs (birds and crocodiles), the testudines (turtles) and the squamates (lizards and snakes).

Direct evidence supporting the existence of a helical dislocation in protofilament packing in the intermediate filaments of oxidized trichocyte keratin.

The X-ray diffraction patterns of quill and hair, as well as other trichocyte keratin appendages, contain meridional reflections that can be indexed on an axial repeat of 470 Å. Unusually, however, many of the expected orders are not observed. A possible explanation, proposed by Fraser and MacRae (1983), was that the intermediate filaments (IF) that constitute the fibrillar component of the filament/matrix texture consist of 4-chain protofilaments arranged on a surface lattice subject to a helical dislocation. The radial projection of the resulting 8-protofilament ribbon was defined in terms of a two-dimensional unit cell characterized by vectors (a, b) with axial projections za ∼ 74 Šand zb ∼ 198 Å. This situation resembles that found in microtubules, where helical dislocations in subunit packing are also encountered, leading to a so-called "seam" along their length (Metoz and Wade, 1997). In keratin, however, the protofilaments are helical so the seam is inclined to the axis of the IF. Here we report details of the Patterson function that provides independent evidence for both the helical dislocation and the dimensions of the surface lattice. In addition, the observed meridional X-ray amplitudes have been compared with those predicted by various models of the axial distribution of electron density. A new model, adapted from one previously proposed, fits the data significantly better than has heretofore proved possible. An interpretation of the model in terms of either specific keratin-associated-protein (KAP) binding or the retention of IF symmetry by a portion of the head and/or tail domains is suggested.

Trichocyte Keratin-Associated Proteins (KAPs).

The trichocyte (hard α-) keratins are epidermal appendages (hair, wool, hoof, horn, claw, baleen and quill) with a classic filament-matrix composite structure. In human hair, for example, keratin intermediate filaments (IF) of diameter 7.5 nm are embedded in a matrix formed from at least 89 different types of keratin-associated proteins (KAPs). The latter fall into three families, generally defined in terms of their cysteine residue or glycine plus tyrosine residue content. The KAPs, which infiltrate the space between the IF, are recognized as having especially important roles in the organisation of the IF into macrofibrils, in determining some of the most important physical attributes of the fully-keratinised hair fibre, including its hardness, toughness and pliability, and in linking IF to one another, either directly or indirectly, with a resultant increase in durability and resistance to degradation by microorganisms. Sequence data for many KAPs are now available, and repeating motifs of varying extent have been observed in a number of them. Little, however, is known about their three-dimensional structures, though modelling has indicated that some local structural regularity is likely to exist. Current data suggest that the KAPs in vivo may adopt a variety of energetically-similar conformations stabilized predominantly by intramolecular disulfide bonds. The role of KAPs in hair diseases relates more to modulation in gene expression than to point mutations, in contrast to that observed for the IF proteins.

Structural Hierarchy of Trichocyte Keratin Intermediate Filaments.

Although trichocyte keratins (hair, wool, quill, claw) have been studied since the 1930s it is only over the last 30 years or so that major advances have been made in our understanding of the complex structural hierarchy of the filamentous component of this important filament-matrix composite. A variety of techniques, including amino acid sequence analysis, computer modelling, X-ray fibre diffraction and protein crystallography, various forms of electron microscopy, and crosslinking methods have now combined to reveal much of the structural detail. The heterodimeric structure of the keratin molecule is clear, as are the highly-specific modes by which these molecules aggregate to form functionally viable IF. The observation that hair keratin can adopt not one but two structurally-distinct conformations, one formed in the living cells at the base of the hair follicle in a reducing environment and the second in the fully differentiated hair in dead cells in an oxidized state, was unexpected but has major implications for the mechanism of hair growth. Insights have also been made into the mechanism of the uppermost level of hair superstructure, relating to the assembly of the IF in the paracortical and orthocortical macrofibrils.

Intermediate filament structure in fully differentiated (oxidised) trichocyte keratin.

For the past 50years there has been considerable debate over the sub-structure of the fully differentiated (oxidised) trichocyte keratin intermediate filament. Depending on the staining and preparative procedures employed, IF observed in transverse section in the transmission electron microscope have varied in appearance between that of a "ring" and a "ring-core" structure, corresponding to the so-called (8+0) and (7+1) protofilament arrangements. In a new analysis of the fine structure of the 1nm equatorial region of the X-ray diffraction pattern of quill we show that the observed pattern is consistent with the (8+0) model and we are also able to assign values to the various parameters. In contrast, we show that the observed X-ray pattern is inconsistent with a (7+1) arrangement. Furthermore, in the (7+1) model steric hindrance would be encountered between the core protofilament and those constituting the ring. The appearance of a central "core" in transverse TEM sections, previously attributed to a central protofilament, is explained in terms of portions of the apolar, disulfide-bonded head and/or tail domains of the trichocyte keratin IF molecules, including the conserved H subdomains, lying along the axis of the IF, thereby decreasing the efficacy of the reducing agents used prior to staining. The H1 subdomain, previously shown to be important in the assembly of epidermal IF molecules at the two- to four-molecule level, is likely to have a similar role for the trichocyte keratins and may form part of a central scaffold on which the molecules assemble into fully functional IF.

Fibrous Protein Structures: Hierarchy, History and Heroes.

During the 1930s and 1940s the technique of X-ray diffraction was applied widely by William Astbury and his colleagues to a number of naturally-occurring fibrous materials. On the basis of the diffraction patterns obtained, he observed that the structure of each of the fibres was dominated by one of a small number of different types of molecular conformation. One group of fibres, known as the k-m-e-f group of proteins (keratin - myosin - epidermin - fibrinogen), gave rise to diffraction characteristics that became known as the α-pattern. Others, such as those from a number of silks, gave rise to a different pattern - the ß-pattern, while connective tissues yielded a third unique set of diffraction characteristics. At the time of Astbury's work, the structures of these materials were unknown, though the spacings of the main X-ray reflections gave an idea of the axial repeats and the lateral packing distances. In a breakthrough in the early 1950s, the basic structures of all of these fibrous proteins were determined. It was found that the long protein chains, composed of strings of amino acids, could be folded up in a systematic manner to generate a limited number of structures that were consistent with the X-ray data. The most important of these were known as the α-helix, the ß-sheet, and the collagen triple helix. These studies provided information about the basic building blocks of all proteins, both fibrous and globular. They did not, however, provide detailed information about how these molecules packed together in three-dimensions to generate the fibres found in vivo. A number of possible packing arrangements were subsequently deduced from the X-ray diffraction and other data, but it is only in the last few years, through the continued improvements of electron microscopy, that the packing details within some fibrous proteins can now be seen directly. Here we outline briefly some of the milestones in fibrous protein structure determination, the role of the amino acid sequences and how new techniques, including electron microscopy, are helping to define fibrous protein structures in three-dimensions. We also introduce the idea that, from the known sequence characteristics of different fibrous proteins, new molecules can be designed and synthesized, thereby generating new biological materials with specific structural properties. Some of these, for example, are planned for use in drug delivery systems. Along the way we also introduce the various Chapters of the book, where individual fibrous proteins are discussed in detail.

Structural Transition of Trichocyte Keratin Intermediate Filaments During Development in the Hair Follicle.

The intermediate filaments (IF) in trichocyte (hard α-) keratin are unique amongst the various classes of IF in having not one but two topologically-distinct structures. The first is formed at an early stage of hair development in a reducing environment within the cells in the lower part of the follicle. The second structure occurs at a later stage of hair development in the upper part of the follicle, where there is a transition to an oxidizing environment. Crosslinking studies reveal that molecular slippage occurs within the IF upon oxidation and that this results in many cysteine residues lying in near axial alignment, thereby facilitating disulphide bond formation. The disulphide bonds so formed stabilize the assembly of IF molecules and convert the keratin fibre into a tough, resilient and insoluble structure suitable for its function in vivo as a thermo-regulator and a protector of the animal against its external environment.

Filamentous Structure of Hard ß-Keratins in the Epidermal Appendages of Birds and Reptiles.

The structures of avian and reptilian epidermal appendages, such as feathers, claws and scales, have been modelled using X-ray diffraction and electron microscopy data, combined with sequence analyses. In most cases, a family of closely related molecules makes up the bulk of the appendage, and each of these molecules contains a central ß-rich 34-residue segment, which has been identified as the principal component of the framework of the 3.4 nm diameter filaments. The N- and C-terminal segments form the matrix component of the filament/matrix complex. The 34-residue ß-rich central domains occur in pairs, related by either a parallel dyad or a perpendicular dyad axis, and form a ß-sandwich stabilized by apolar interactions. They are also twisted in a right-handed manner. In feather, the filaments are packed into small sheets and it is possible to determine their likely orientation within the sheets from the low-angle X-ray diffraction data. The physical properties of the various epidermal appendages can be related to the amino acid sequence and composition of defined molecular segments characteristic of the chains concerned.