Zebrafish as a model for cardiac disease; Cryo-EM structure of native cardiac thin filaments from Danio Rerio.

Actin, tropomyosin and troponin, the proteins that comprise the contractile apparatus of the cardiac thin filament, are highly conserved across species. We have used cryo-EM to study the three-dimensional structure of the zebrafish cardiac thin and actin filaments. With 70% of human genes having an obvious zebrafish orthologue, and conservation of 85% of disease-causing genes, zebrafish are a good animal model for the study of human disease. Our structure of the zebrafish thin filament reveals the molecular interactions between the constituent proteins, showing that the fundamental organisation of the complex is the same as that reported in the human reconstituted thin filament. A reconstruction of zebrafish cardiac F-actin demonstrates no deviations from human cardiac actin over an extended length of 14 actin subunits. Modelling zebrafish homology models into our maps enabled us to compare, in detail, the similarity with human models. The structural similarities of troponin-T in particular, a region known to contain a hypertrophic cardiomyopathy 'hotspot', confirm the suitability of zebrafish to study these disease-causing mutations.

In Vivo Characterization of Endogenous Cardiovascular Extracellular Vesicles in Larval and Adult Zebrafish.

Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen.

Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organization to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small molecule-induced, microtubule-based, cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in situ study of microtubule structure and contents.

After the revolution: how is Cryo-EM contributing to muscle research?

The technique of electron microscopy (EM) has been fundamental to muscle research since the days of Huxley and Hanson. Direct observation of how proteins in the sarcomere are arranged and visualising the changes that occur upon activation have greatly increased our understanding of function. In the 1980s specimen preparation techniques for biological EM moved away from traditional fixing and staining. The technique known as cryo-electron microscopy (Cryo-EM) was developed, which involves rapidly freezing proteins in liquid ethane which maintains them in a near native state. Within the last 5 years there has been a step change in the achievable resolution using Cryo-EM. This 'resolution revolution' can be attributed to advances in detector technology, microscope automation and maximum likelihood image processing. In this article we look at how Cryo-EM has contributed to the field of muscle research in this post revolution era, focussing on recently published high resolution structures of sarcomeric proteins.

Relaxed and active thin filament structures; a new structural basis for the regulatory mechanism.

The structures of muscle thin filaments reconstituted using skeletal actin and cardiac troponin and tropomyosin have been determined with and without bound Ca2+ using electron microscopy and reference-free single particle analysis. The resulting density maps have been fitted with atomic models of actin, tropomyosin and troponin showing that: (i) the polarity of the troponin complex is consistent with our 2009 findings, with large shape changes in troponin between the two states; (ii) without Ca2+ the tropomyosin pseudo-repeats all lie at almost equivalent positions in the 'blocked' position on actin (over subdomains 1 and 2); (iii) in the active state the tropomyosin pseudo-repeats are all displaced towards subdomains 3 and 4 of actin, but the extent of displacement varies within the regulatory unit depending upon the axial location of the pseudo-repeats with respect to troponin. Individual pseudo-repeats with Ca2+ bound to troponin can be assigned either to the 'closed' state, a partly activated conformation, or the 'M-state', a fully activated conformation which has previously been thought to occur only when myosin heads bind. These results lead to a modified view of the steric blocking model of thin filament regulation in which cooperative activation is governed by troponin-mediated local interactions of the pseudo-repeats of tropomyosin with actin.

Myosin and Actin Filaments in Muscle: Structures and Interactions.

In the last decade, improvements in electron microscopy and image processing have permitted significantly higher resolutions to be achieved (sometimes <1 nm) when studying isolated actin and myosin filaments. In the case of actin filaments the changing structure when troponin binds calcium ions can be followed using electron microscopy and single particle analysis to reveal what happens on each of the seven non-equivalent pseudo-repeats of the tropomyosin α-helical coiled-coil. In the case of the known family of myosin filaments not only are the myosin head arrangements under relaxing conditions being defined, but the latest analysis, also using single particle methods, is starting to reveal the way that the α-helical coiled-coil myosin rods are packed to give the filament backbones.

Internal (His)6-tagging delivers a fully functional hetero-oligomeric class II chaperonin in high yield.

Group II chaperonins are ATP-ases indispensable for the folding of many proteins that play a crucial role in Archaea and Eukarya. They display a conserved two-ringed assembly enclosing an internal chamber where newly translated or misfolded polypeptides can fold to their native structure. They are mainly hexadecamers, with each eight-membered ring composed of one or two (in Archaea) or eight (in Eukarya) different subunits. A major recurring problem within group II chaperonin research, especially with the hetero-oligomeric forms, is to establish an efficient recombinant system for the expression of large amounts of wild-type as well as mutated variants. Herein we show how we can produce, in E. coli cells, unprecedented amounts of correctly assembled and active αß-thermosome, the class II chaperonin from Thermoplasma acidophilum, by introducing a (His)6-tag within a loop in the α subunit of the complex. The specific location was identified via a rational approach and proved not to disturb the structure of the chaperonin, as demonstrated by size-exclusion chromatography, native gel electrophoresis and electron microscopy. Likewise, the tagged protein showed an ATP-ase activity and an ability to refold substrates identical to the wild type. This tagging strategy might be employed for the overexpression of other recombinant chaperonins.

Effects of unweighting and speed on in-shoe regional loading during running on a lower body positive pressure treadmill.

The purpose of this study was to determine how unweighted running on a lower body positive pressure treadmill (LBPPT) modifies in-shoe regional loading. Ten experienced runners were fit with pressure distribution measurement insoles and ran at 100%, 120%, and 140% of self-reported easy training pace on a LBPPT at 20%, 40%, 60%, 80%, and 100% body weight percentage settings (BWSet). Speeds and BWSet were in random order. A linear mixed effect model (p<0.05 significance level) was used to compare differences in whole foot and regional maximum in-shoe plantar force (FMAX), impulse, and relative load distribution across speeds and BWSet. There were significant main effects (p<0.001) for running speed and BWSet for whole foot Fmax and impulse. The model revealed 1.4% and 0.24% increases in whole foot FMAX (times body weight) and impulse, respectively, for every unit increase in body weight percentage. There was a significant main effect for BWSet on Fmax and relative load (p<0.05) for each of the nine foot regions examined, though four regions were not different between 80% and 100% BWSet. There was a significant (p<0.001) main effect for BWSet on forefoot to rear foot relative load. Linear relationships were found between increases in BWSet and increases in-shoe Fmax and impulse, resulting from regional changes in foot pressure which represent a shift towards forefoot loading, most evident <80% BWSet. Estimating in-shoe regional loading parameters may be useful during rehabilitation and training to appropriately prescribe specific speed and body weight levels, without exceeding certain critical peak force levels while running.

Radiological variables associated with progression of femoroacetabular impingement of the hip: a systematic review.

OBJECTIVES: Femoroacetabular impingement is gaining increased recognition as a cause of hip dysfunction. Of great concern is its potential association with labral tears and osteoarthritis. This systematic review examines the evidence regarding radiographic variables associated with the progression of femoroacetabular impingement. DESIGN: Systematic review. METHODS: Articles were selected following a comprehensive search of PubMed, CINAHL, SportDiscus, Embase, and Medline databases from database inception through October 2012. Inclusion criteria involved (1) estimates of the association between prognostic variables and progression of femoroacetabular impingement, (2) prospective or retrospective design, (3) patients diagnosed with femoroacetabular impingement based on established criteria, (4) the outcome of interest was radiologic and/or clinical progression of femoroacetabular impingement, and (5) access to the full text. Two independent reviewers assessed the methodological quality of each study and the association between prognostic variables and femoroacetabular impingement progression. RESULTS: Thirteen articles met the inclusion criteria; nine were considered to be of high quality. Moderate evidence of progression of femoroacetabular impingement to labral pathology was associated with increased alpha angle. Moderate evidence for their lack of association with progression of FAI was associated with alpha angle with respect to development of osteoarthritis, acetabular index, center edge angle, coxa profunda, coxa vara, and pistol grip deformity. CONCLUSIONS: There is moderate evidence that increased alpha angle at baseline is associated with progression of femoroacetabular impingement to labral tear. Moderate evidence suggests a lack of association between other radiographic variables and progression of femoroacetabular impingement.

A novel approach to the structural analysis of partially decorated actin based filaments.

We describe a novel set of single particle based procedures for the structural analysis of electron microscope images of muscle thin filaments and other partially decorated actin based filaments. The thin filament comprises actin and the regulatory proteins tropomyosin and troponin in a 7:1:1M ratio. Prior to our work, structure analysis from electron microscope images of the thin filament has largely involved either helical averaging defined by the underlying actin helix or the use of single particle analysis but using a starting model as a reference structure. Our single particle based approach yields an accurate structure for the complete thin filament by avoiding the loss of information from troponin and tropomyosin associated with helical averaging and also removing the potential reference bias associated with the use of a starting model. The approach is more widely applicable to sub-stoichiometric complexes of F-actin and actin-binding proteins.

Structure and orientation of troponin in the thin filament.

The troponin complex on the thin filament plays a crucial role in the regulation of muscle contraction. However, the precise location of troponin relative to actin and tropomyosin remains uncertain. We have developed a method of reconstructing thin filaments using single particle analysis that does not impose the helical symmetry of actin and is independent of a starting model. We present a single particle three-dimensional reconstruction of the thin filament. Atomic models of the F-actin filament were fitted into the electron density maps and troponin and tropomyosin located. The structure provides evidence that the globular head region of troponin labels the two strands of actin with a 27.5-A axial stagger. The density attributed to troponin appears tapered with the widest point toward the barbed end. This leads us to interpret the polarity of the troponin complex in the thin filament as reversed with respect to the widely accepted model.

A measure for the angle between projections based on the extent of correlation between corresponding central sections.

A pre-condition for the ab initio assignment of Euler angles to a set of projections from an asymmetric object is that at least three of the available projections correspond to rotations about different axes. For symmetric objects this condition may be relaxed. There are some applications of single-particle electron microscopy, such as the reconstruction of filamentous macromolecular assemblies, where all available projections more-or-less correspond to rotations about a common rotation axis making it difficult to satisfy this condition. Here, a method has been developed to overcome this problem, based on the fact that the correlation between two central sections of the Fourier transform of a compact object will not be limited to an infinitesimal central line but will have a finite extent, which is related to the angle between the corresponding projections. Projections from model filaments, with different degrees of rotational symmetry about the long axis, have been used to test the methodology. The results show that angle determination is robust down to signal-to-noise ratios as low as 2 and that, in general, the error decreases as the degree of symmetry increases. The method has been used to assign angles to a set of negatively stained muscle thick filament projections to obtain an initial 3D reconstruction. The main features of the projections are seen to be faithfully reproduced in the reprojections from the reconstruction. A real-space adaptation of this method is also discussed.

Single particle analysis of filamentous and highly elongated macromolecular assemblies.

The application of single particle techniques to the three-dimensional analysis of electron microscope images of elongated or filamentous macromolecular assemblies is evaluated, taking as an example the muscle thin filament. Although the thin filament contains local helical symmetry, because of the inherent variable twist along it, the helical coherence does not extend for large enough distances to allow the symmetry to be used for full reconstruction of the tropomyosin/troponin repeat along the filament. The muscle thin filament therefore represents a general case of a filamentous object in that it is not possible to exploit symmetry in a full analysis. Due to the nature of the imaging process in the electron microscope, only projections of the thin filament around its long axis are available without tilting the grid. Crucially, projection images around a single axis do not provide enough information to assign Euler angles ab initio using current methods. Tests with a model thin filament structure indicated that an out-of-plane tilt of approximately 20 degrees was needed for ab initio angular assignment of sufficient accuracy to calculate a 3D structure to a resolution of approximately 25 A. If no out-of-plane views are available, an alternative approach is to use a prior 3D model as a reference for the initial angle assignment. Tests with the thin filament model indicated that reasonably accurate angular assignment can be made using a reference containing actin, but lacking the regulatory proteins tropomyosin and troponin. We also found that an adaptation of the exact filtered back projection method is required to allow the correct weighting of projection images in which the particle has a very large axial ratio. This adaptation resulted in significant improvements in the reconstruction.