Membrane structures and functional correlates in the bi-segmented eye lens of the cephalopod.

The cephalopod eye lens is unique because it has evolved as a compound structure with two physiologically distinct segments. However, the detailed ultrastructure of this lens and precise optical role of each segment are far from clear. To help elucidate structure-function relationships in the cephalopod lens, we conducted multiple structural investigations on squid. Synchrotron x-ray scattering and transmission electron microscopy disclose that an extensive network of structural features that resemble cell membrane complexes form a substantial component of both anterior and posterior lens segments. Optically, the segments are distinct, however, and Talbot interferometry indicates that the posterior segment possesses a noticeably higher refractive index gradient. We propose that the hitherto unrecognised network of membrane structures in the cephalopod lens has evolved to act as an essential conduit for the internal passage of ions and other metabolic agents through what is otherwise a highly dense structure owing to a very high protein concentration.

Structural control of corneal transparency, refractive power and dynamics.

The cornea needs to be transparent to visible light and precisely curved to provide the correct refractive power. Both properties are governed by its structure. Corneal transparency arises from constructive interference of visible light due to the relatively ordered arrangement of collagen fibrils in the corneal stroma. The arrangement is controlled by the negatively charged proteoglycans surrounding the fibrils. Small changes in fibril organisation can be tolerated but larger changes cause light scattering. Corneal keratocytes do not scatter light because their refractive index matches that of the surrounding matrix. When activated, however, they become fibroblasts that have a lower refractive index. Modelling shows that this change in refractive index significantly increases light scatter. At the microscopic level, the corneal stroma has a lamellar structure, the parallel collagen fibrils within each lamella making a large angle with those of adjacent lamellae. X-ray scattering has shown that the lamellae have preferred orientations in the human cornea: inferior-superior and nasal-temporal in the central cornea and circumferential at the limbus. The directions at the centre of the cornea may help withstand the pull of the extraocular muscles whereas the pseudo-circular arrangement at the limbus supports the change in curvature between the cornea and sclera. Elastic fibres are also present; in the limbus they contain fibrillin microfibrils surrounding an elastin core, whereas at the centre of the cornea, they exist as thin bundles of fibrillin-rich microfibrils. We present a model based on the structure described above that may explain how the cornea withstands repeated pressure changes due to the ocular pulse.

Analysis methods and quality criteria for investigating muscle physiology using x-ray diffraction.

X-ray diffraction studies of muscle have been tremendously powerful in providing fundamental insights into the structures of, for example, the myosin and actin filaments in a variety of muscles and the physiology of the cross-bridge mechanism during the contractile cycle. However, interpretation of x-ray diffraction patterns is far from trivial, and if modeling of the observed diffraction intensities is required it needs to be performed carefully with full knowledge of the possible pitfalls. Here, we discuss (1) how x-ray diffraction can be used as a tool to monitor various specific muscle properties and (2) how to get the most out of the rest of the observed muscle x-ray diffraction patterns by modeling where the reliability of the modeling conclusions can be objectively tested. In other x-ray diffraction methods, such as protein crystallography, the reliability of every step of the process is estimated and quoted in published papers. In this way, the quality of the structure determination can be properly assessed. To be honest with ourselves in the muscle field, we need to do as near to the same as we can, within the limitations of the techniques that we are using. We discuss how this can be done. We also use test cases to reveal the dos and don'ts of using x-ray diffraction to study muscle physiology.

The muscle M3 x-ray diffraction peak and sarcomere length: No evidence for disordered myosin heads out of actin overlap.

X-ray diffraction studies of muscle have provided a wealth of information on muscle structure and physiology, and the meridian of the diffraction pattern is particularly informative. Reconditi et al. (2014. J. Physiol.https://doi.org/10.1113/jphysiol.2013.267849) performed superb experiments on changes to the M3 meridional peak as a function of sarcomere length (SL). They found that the M3 intensity dropped almost linearly as sarcomere length increased at least to about SL = 3.0 µm, and that it followed the same track as tension, pointing toward zero at the end of overlap at ∼3.6 µm. They concluded that, just as tension could only be generated by overlapped myosin heads, so ordered myosin heads contributing to the M3 intensity could only occur in the overlap region of the A-band, and that nonoverlapped heads must be highly disordered. Here we show that this conclusion is not consistent with x-ray diffraction theory; it would not explain their observations. We discuss one possible reason for the change in M3 intensity with increasing sarcomere length in terms of increasing axial misalignment of the myosin filaments that at longer sarcomere lengths is limited by the elastic stretching of the M-band and titin.

The Transient Mechanics of Muscle Require Only a Single Force-Producing Cross-Bridge State and a 100 Å Working Stroke.

An informative probe of myosin cross-bridge behaviour in active muscle is a mechanical transient experiment where, for example, a fully active muscle initially held at constant length is suddenly shortened to a new fixed length, providing a force transient, or has its load suddenly reduced, providing a length transient. We describe the simplest cross-bridge mechanical cycle we could find to model these transients. We show using the statistical mechanics of 50,000 cross-bridges that a simple cycle with two actin-attached cross-bridge states, one producing no force and the other producing force, will explain much of what has been observed experimentally, and we discuss the implications of this modelling for our understanding of how muscle works. We show that this same simple model will explain, reasonably well, the isotonic mechanical and X-ray transients under different loads observed by Reconditi et al. (2004, Nature 428, 578) and that there is no need to invoke different cross-bridge step sizes under these different conditions; a step size of 100 Å works well for all loads. We do not claim that this model provides a total mechanical explanation of how muscle works. However, we do suggest that only if there are other observations that cannot be explained by this simple model should something more complicated be considered.

Ultrastructural variability of the juxtacanalicular tissue along the inner wall of Schlemm's canal.

Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm's canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet. In the present study, regional variations in the ultrastructure of the JCT and the inner wall of SC were investigated in three dimensions. Methods: With the use of serial block face scanning electron microscopy (SBF-SEM), the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix was investigated and quantified. The distribution of giant vacuoles (GVs) and pores in the inner wall endothelium of SC was further examined. Results: With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). Circumferential variability in the type and distribution of GVs was observed, and the percentage of GVs with pores varied between 3% and 27%. Conclusions: These studies provide a detailed quantitative analysis of the ultrastructure of JCT and the distribution of GVs along the circumference of SC in three dimensions, supporting the non-uniform or segmental aqueous outflow.

Myosin Cross-Bridge Behaviour in Contracting Muscle-The T1 Curve of Huxley and Simmons (1971) Revisited.

The stiffness of the myosin cross-bridges is a key factor in analysing possible scenarios to explain myosin head changes during force generation in active muscles. The seminal study of Huxley and Simmons (1971: Nature 233: 533) suggested that most of the observed half-sarcomere instantaneous compliance (=1/stiffness) resides in the myosin heads. They showed with a so-called T1 plot that, after a very fast release, the half-sarcomere tension reduced to zero after a step size of about 60Å (later with improved experiments reduced to 40Å). However, later X-ray diffraction studies showed that myosin and actin filaments themselves stretch slightly under tension, which means that most (at least two-thirds) of the half sarcomere compliance comes from the filaments and not from cross-bridges. Here we have used a different approach, namely to model the compliances in a virtual half sarcomere structure in silico. We confirm that the T1 curve comes almost entirely from length changes in the myosin and actin filaments, because the calculated cross-bridge stiffness (probably greater than 0.4 pN/Å) is higher than previous studies have suggested. Our model demonstrates that the formulations produced by previous authors give very similar results to our model if the same starting parameters are used. However, we find that it is necessary to model the X-ray diffraction data as well as mechanics data to get a reliable estimate of the cross-bridge stiffness. In the light of the high cross-bridge stiffness found in the present study, we present a plausible modified scenario to describe aspects of the myosin cross-bridge cycle in active muscle. In particular, we suggest that, apart from the filament compliances, most of the cross-bridge contribution to the instantaneous T1 response may come from weakly-bound myosin heads, not myosin heads in strongly attached states. The strongly attached heads would still contribute to the T1 curve, but only in a very minor way, with a stiffness that we postulate could be around 0.1 pN/Å, a value which would generate a working stroke close to 100 Å from the hydrolysis of one ATP molecule. The new model can serve as a tool to calculate sarcomere elastic properties for any vertebrate striated muscle once various parameters have been determined (e.g., tension, T1 intercept, temperature, X-ray diffraction spacing results).

The Interacting Head Motif Structure Does Not Explain the X-Ray Diffraction Patterns in Relaxed Vertebrate (Bony Fish) Skeletal Muscle and Insect (Lethocerus) Flight Muscle.

Unlike electron microscopy, which can achieve very high resolution but to date can only be used to study static structures, time-resolved X-ray diffraction from contracting muscles can, in principle, be used to follow the molecular movements involved in force generation on a millisecond timescale, albeit at moderate resolution. However, previous X-ray diffraction studies of resting muscles have come up with structures for the head arrangements in resting myosin filaments that are different from the apparently ubiquitous interacting head motif (IHM) structures found by single particle analysis of electron micrographs of isolated myosin filaments from a variety of muscle types. This head organization is supposed to represent the super-relaxed state of the myosin filaments where adenosine triphosphate (ATP) usage is minimized. Here we have tested whether the interacting head motif structures will satisfactorily explain the observed low-angle X-ray diffraction patterns from resting vertebrate (bony fish) and invertebrate (insect flight) muscles. We find that the interacting head motif does not, in fact, explain what is observed. Previous X-ray models fit the observations much better. We conclude that the X-ray diffraction evidence has been well interpreted in the past and that there is more than one ordered myosin head state in resting muscle. There is, therefore, no reason to question some of the previous X-ray diffraction results on myosin filaments; time-resolved X-ray diffraction should be a reliable way to follow crossbridge action in active muscle and may be one of the few ways to visualise the molecular changes in myosin heads on a millisecond timescale as force is actually produced.

Cell-independent matrix configuration in early corneal development.

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3.

Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle.

Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres.

Monitoring the myosin crossbridge cycle in contracting muscle: steps towards 'Muscle-the Movie'.

Some vertebrate muscles (e.g. those in bony fish) have a simple lattice A-band which is so well ordered that low-angle X-ray diffraction patterns are sampled in a simple way amenable to crystallographic techniques. Time-resolved X-ray diffraction through the contractile cycle should provide a movie of the molecular movements involved in muscle contraction. Generation of 'Muscle-The Movie' was suggested in the 1990s and since then efforts have been made to work out how to achieve it. Here we discuss how a movie can be generated, we discuss the problems and opportunities, and present some new observations. Low angle X-ray diffraction patterns from bony fish muscles show myosin layer lines that are well sampled on row-lines expected from the simple hexagonal A-band lattice. The 1st, 2nd and 3rd myosin layer lines at d-spacings of around 42.9 nm, 21.5 nm and 14.3 nm respectively, get weaker in patterns from active muscle, but there is a well-sampled intensity remnant along the layer lines. We show here that the pattern from the tetanus plateau is not a residual resting pattern from fibres that have not been fully activated, but is a different well-sampled pattern showing the presence of a second, myosin-centred, arrangement of crossbridges within the active crossbridge population. We also show that the meridional M3 peak from active muscle has two components of different radial widths consistent with (i) active myosin-centred (probably weak-binding) heads giving a narrow peak and (ii) heads on actin in strong states giving a broad peak.

Objective Derivation of the Morphology and Staging of Visual Field Loss Associated with Long-Term Vigabatrin Therapy.

BACKGROUND: The morphology and between-eye symmetry of the visual field loss associated with the antiepileptic drug vigabatrin (VAVFL) has received little attention. OBJECTIVE: Our objective was to model the appearance and ensuing staging of VAVFL derived with the European Medicines Agency-approved perimetric protocol. METHODS: This was a retrospective, cross-sectional, observational study that identified 123 adults who had received vigabatrin for refractory seizures and who had no evidence of co-existing retino-geniculo-cortical visual pathway abnormality. A further 38 adults with refractory seizures and identical inclusion criteria but no exposure to vigabatrin acted as controls. For each group, the median outcome at each stimulus location in each eye (of absolute loss, relative loss or Pattern Deviation probability level, as appropriate) was derived for each successive ten pairs of fields, ranked for severity. Between-eye symmetry was quantified by an index that accounted for severity of loss and that was referenced to the likelihood of the occurrence of symmetry due to chance. RESULTS: The modelled VAVFL was bilateral and highly symmetrical and was described by six stages that were all independent of the extent of vigabatrin exposure. The loss originated in the extreme temporal periphery and encroached centripetally along all meridians towards fixation. The initial appearance within the central field (Stage 2) occurred inferior-nasally. Subsequent stages exhibited increasing loss, which was greater nasally than temporally. Stage 6 described concentric loss extending to approximately 15° eccentricity from fixation. CONCLUSION: The model exhibited a consistent pattern of VAVFL. The staging of the loss could assist the risk:benefit analysis of vigabatrin for the treatment of epilepsy.

The Topographical Relationship between Visual Field Loss and Peripapillary Retinal Nerve Fibre Layer Thinning Arising from Long-Term Exposure to Vigabatrin.

BACKGROUND: The antiepileptic drug vigabatrin is associated with characteristic visual field loss (VAVFL) and thinning of the peripapillary retinal nerve fibre layer (PPRNFL); however, the relationship is equivocal. OBJECTIVE: The aim of this study was to determine the function-structure relationship associated with long-term exposure to vigabatrin, thereby improving the risk/benefit analysis of the drug. METHODS: A cross-sectional observational design identified 40 adults who had received long-term vigabatrin for refractory seizures, who had no evidence of co-existing retino-geniculo-cortical visual pathway abnormality, and who had undergone a standardized protocol of perimetry and of optical coherence tomography (OCT) of the PPRNFL. Vigabatrin toxicity was defined as the presence of VAVFL. The function-structure relationship for the superior and inferior retinal quadrants was evaluated by two established models applicable to other optic neuropathies. RESULTS: The function-structure relationship for each model was consistent with an optic neuropathy. PPRNFL thinning, expressed in micrometres, asymptoted at an equivalent visual field loss of worse than approximately - 10.0 dB, thereby preventing assessment of more substantial thinning. Transformation of the outcomes to retinal ganglion cell soma and axon estimates, respectively, resulted in a linear relationship. CONCLUSIONS: Functional and structural abnormality is strongly related in individuals with vigabatrin toxicity and no evidence of visual pathway comorbidity, thereby implicating retinal ganglion cell dysfunction. OCT affords a limited measurement range compared with perimetry: severity cannot be directly assessed when the PPRNFL quadrant thickness is less than approximately 65 µm, depending on the tomographer. This limitation can be overcome by transformation of thickness to remaining axons, an outcome requiring input from perimetry.

Different Myosin Head Conformations in Bony Fish Muscles Put into Rigor at Different Sarcomere Lengths.

At a resting sarcomere length of approximately 2.2 µm bony fish muscles put into rigor in the presence of BDM (2,3-butanedione monoxime) to reduce rigor tension generation show the normal arrangement of myosin head interactions with actin filaments as monitored by low-angle X-ray diffraction. However, if the muscles are put into rigor using the same protocol but stretched to 2.5 µm sarcomere length, a markedly different structure is observed. The X-ray diffraction pattern is not just a weaker version of the pattern at full overlap, as might be expected, but it is quite different. It is compatible with the actin-attached myosin heads being in a different conformation on actin, with the average centre of cross-bridge mass at a higher radius than in normal rigor and the myosin lever arms conforming less to the actin filament geometry, probably pointing back to their origins on their parent myosin filaments. The possible nature of this new rigor cross-bridge conformation is discussed in terms of other well-known states such as the weak binding state and the 'roll and lock' mechanism; we speculate that we may have trapped most myosin heads in an early attached strong actin-binding state in the cross-bridge cycle on actin.

X-ray Diffraction Evidence for Low Force Actin-Attached and Rigor-Like Cross-Bridges in the Contractile Cycle.

Defining the structural changes involved in the myosin cross-bridge cycle on actin in active muscle by X-ray diffraction will involve recording of the whole two dimensional (2D) X-ray diffraction pattern from active muscle in a time-resolved manner. Bony fish muscle is the most highly ordered vertebrate striated muscle to study. With partial sarcomere length (SL) control we show that changes in the fish muscle equatorial A-band (10) and (11) reflections, along with (10)/(11) intensity ratio and the tension, are much more rapid than without such control. Times to 50% change with SL control were 19.5 (±2.0) ms, 17.0 (±1.1) ms, 13.9 (±0.4) ms and 22.5 (±0.8) ms, respectively, compared to 25.0 (±3.4) ms, 20.5 (±2.6) ms, 15.4 (±0.6) ms and 33.8 (±0.6) ms without control. The (11) intensity and the (10)/(11) intensity ratio both still change ahead of tension, supporting the likelihood of the presence of a head population close to or on actin, but producing little or no force, in the early stages of the contractile cycle. Higher order equatorials (e.g., (30), (31), and (32)), more sensitive to crossbridge conformation and distribution, also change very rapidly and overshoot their tension plateau values by a factor of around two, well before the tension plateau has been reached, once again indicating an early low-force cross-bridge state in the contractile cycle. Modelling of these intensity changes suggests the presence of probably two different actin-attached myosin head structural states (mainly low-force attached and rigor-like). No more than two main attached structural states are necessary and sufficient to explain the observations. We find that 48% of the heads are off actin giving a resting diffraction pattern, 20% of heads are in the weak binding conformation and 32% of the heads are in the strong (rigor-like) state. The strong states account for 96% of the tension at the tetanus plateau.

Role of Decorin Core Protein in Collagen Organisation in Congenital Stromal Corneal Dystrophy (CSCD).

The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD). In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT) in the decorin (DCN) gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS), to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.

Measuring the Refractive Index of Bovine Corneal Stromal Cells Using Quantitative Phase Imaging.

The cornea is the primary refractive lens in the eye and transmits >90% of incident visible light. It has been suggested that the development of postoperative corneal haze could be due to an increase in light scattering from activated corneal stromal cells. Quiescent keratocytes are thought to produce crystallins that match the refractive index of their cytoplasm to the surrounding extracellular material, reducing the amount of light scattering. To test this, we measured the refractive index (RI) of bovine corneal stromal cells, using quantitative phase imaging of live cells in vitro, together with confocal microscopy. The RI of quiescent keratocytes (RI = 1.381 ± 0.004) matched the surrounding matrix, thus supporting the hypothesis that keratocyte cytoplasm does not scatter light in the normal cornea. We also observed that the RI drops after keratocyte activation (RI = 1.365 ± 0.003), leading to a mismatch with the surrounding intercellular matrix. Theoretical scattering models showed that this mismatch would reduce light transmission in the cornea. We conclude that corneal transparency depends on the matching of refractive indices between quiescent keratocytes and the surrounding tissue, and that after surgery or wounding, the resulting RI mismatch between the activated cells and their surrounds significantly contributes to light scattering.

Corneal structure and transparency.

The corneal stroma plays several pivotal roles within the eye. Optically, it is the main refracting lens and thus has to combine almost perfect transmission of visible light with precise shape, in order to focus incoming light. Furthermore, mechanically it has to be extremely tough to protect the inner contents of the eye. These functions are governed by its structure at all hierarchical levels. The basic principles of corneal structure and transparency have been known for some time, but in recent years X-ray scattering and other methods have revealed that the details of this structure are far more complex than previously thought and that the intricacy of the arrangement of the collagenous lamellae provides the shape and the mechanical properties of the tissue. At the molecular level, modern technologies and theoretical modelling have started to explain exactly how the collagen fibrils are arranged within the stromal lamellae and how proteoglycans maintain this ultrastructure. In this review we describe the current state of knowledge about the three-dimensional stromal architecture at the microscopic level, and about the control mechanisms at the nanoscopic level that lead to optical transparency.

Three-dimensional architecture of collagen type VI in the human trabecular meshwork.

PURPOSE: Type VI collagen is a primary component of the extracellular matrix of many connective tissues. It can form distinct aggregates depending on tissue structure, chemical environment, and physiology. In the current study we examine the ultrastructure and mode of aggregation of type VI collagen molecules in the human trabecular meshwork. METHODS: Trabecular meshwork was dissected from donor human eyes, and three-dimensional transmission electron microscopy of type VI collagen aggregates was performed. RESULTS: Electron-dense collagen structures were detected in the human trabecular meshwork and identified as collagen type VI assemblies based on the three-dimensional spatial arrangement of the type VI collagen molecules, the 105-nm axial periodicity of the assemblies themselves, and their characteristic double bands, which arose from the globular domains of the type VI collagen molecules. Sulfated proteoglycans were also seen to associate with the assemblies either with the globular domain or the inner rod-like segments of the tetramers. CONCLUSIONS: No extended structural regularity in the organization of type VI collagen assemblies within the trabecular meshwork was evident, and the lateral separation of the tetramers forming the assemblies varied, as did the angle formed by the main axes of adjacent tetramers. This is potentially reflective of the specific nature of the trabecular meshwork environment, which facilitates aqueous outflow from the eye, and we speculate that extracellular matrix ions and proteins might prevent a more tight packing of type VI collagen tetramers that form the assemblies.

Resolution of the three dimensional structure of components of the glomerular filtration barrier.

BACKGROUND: The human glomerulus is the primary filtration unit of the kidney, and contains the Glomerular Filtration Barrier (GFB). The GFB had been thought to comprise 3 layers - the endothelium, the basement membrane and the podocyte foot processes. However, recent studies have suggested that at least two additional layers contribute to the function of the GFB, the endothelial glycocalyx on the vascular side, and the sub-podocyte space on the urinary side. To investigate the structure of these additional layers is difficult as it requires three-dimensional reconstruction of delicate sub-microscopic (<1 µm) cellular and extracellular elements. METHODS: Here we have combined three different advanced electron microscopic techniques that cover multiple orders of magnitude of volume sampled, with a novel staining methodology (Lanthanum Dysprosium Glycosaminoglycan adhesion, or LaDy GAGa), to determine the structural basis of these two additional layers. Serial Block Face Scanning Electron Microscopy (SBF-SEM) was used to generate a 3-D image stack with a volume of a 5.3 x 105 µm3 volume of a whole kidney glomerulus (13% of glomerular volume). Secondly, Focused Ion Beam milling Scanning Electron Microscopy (FIB-SEM) was used to image a filtration region (48 µm3 volume). Lastly Transmission Electron Tomography (Tom-TEM) was performed on a 0.3 µm3 volume to identify the fine structure of the glycocalyx. RESULTS: Tom-TEM clearly showed 20 nm fibre spacing in the glycocalyx, within a limited field of view. FIB-SEM demonstrated, in a far greater field of view, how the glycocalyx structure related to fenestrations and the filtration slits, though without the resolution of TomTEM. SBF-SEM was able to determine the extent of the sub-podocyte space and glycocalyx coverage, without additional heavy metal staining. Neither SBF- nor FIB-SEM suffered the anisotropic shrinkage under the electron beam that is seen with Tom-TEM. CONCLUSIONS: These images demonstrate that the three dimensional structure of the GFB can be imaged, and investigated from the whole glomerulus to the fine structure of the glycocalyx using three dimensional electron microscopy techniques. This should allow the identification of structural features regulating physiology, and their disruption in pathological states, aiding the understanding of kidney disease.