Christian Pinali

Structural interactions between collagen and proteoglycans are elucidated by three-dimensional electron tomography of bovine cornea

Interactions between collagens and proteoglycans help define the structure and function of extracellular matrices. The cornea, which contains proteoglycans with keratan sulphate or chondroitin/dermatan sulphate glycosaminoglycan chains, is an excellent model system in which to study collagen-proteoglycan structures and interactions. Here, we present the first three-dimensional electron microscopic reconstructions of the cornea, and these include corneas from which glycosaminoglycans have been selectively removed by enzymatic digestion. Our reconstructions show that narrow collagen fibrils associate with sulphated proteoglycans that appear as extended, variable-length linear structures. The proteoglycan network appears to tether two or more collagen fibrils, and thus organize the matrix with enough spatial specificity to fulfill the requirements for corneal transparency. Based on the data, we propose that the characteristic pseudohexagonal fibril arrangement in cornea is controlled by the balance of a repulsive force arising from osmotic pressure and an attractive force due to the thermal motion of the proteoglycans.

The Architecture of the Cornea and Structural Basis of Its Transparency

The cornea is the transparent connective tissue window at the front of the eye. In the extracellular matrix of the corneal stroma, hybrid type I/V collagen fibrils are remarkably uniform in diameter at approximately 30 nm and are regularly arranged into a pseudolattice. Fibrils are believed to be kept at defined distances by the influence of proteoglycans. Light entering the cornea is scattered by the collagen fibrils, but their spatial distribution is such that the scattered light interferes destructively in all directions except from the forward direction. In this way, light travels forward through the cornea to reach the retina. In this chapter, we will review the macromolecular components of the corneal stroma, the way they are organized into a stacked lamellar array, and how this organization guarantees corneal transparency.

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-Tubules This Organization Is Perturbed in Heart Failure

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-Tubules

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

Three-dimensional aspects of matrix assembly by cells in the developing cornea

Significance The cornea is a specialized connective tissue assembled as a remarkably ordered array of superimposed collagenous lamellae, and their component collagen fibrils, essential for optical transparency. Surprisingly, the mechanisms involved in deposition of this unique structure are still not fully understood. Here we have used correlative microscopy techniques, including innovative methods of serial block face scanning electron microscopy, to observe the sequence of corneal matrix formation in three-dimensional reconstructions of embryonic chick cornea. Our data show that corneal cells, keratocytes, exhibit long-range associations with collagen bundles in the developing matrix via an extended network of actin-rich tubular cytoplasmic protrusions, which we term keratopodia. Synchronized alignment of keratopodia and collagen is evident during the course of lamella formation. Cell-directed deposition of aligned collagen fibrils during corneal embryogenesis is poorly understood, despite the fact that it is the basis for the formation of a corneal stroma that must be transparent to visible light and biomechanically stable. Previous studies of the structural development of the specialized matrix in the cornea have been restricted to examinations of tissue sections by conventional light or electron microscopy. Here, we use volume scanning electron microscopy, with sequential removal of ultrathin surface tissue sections achieved either by ablation with a focused ion beam or by serial block face diamond knife microtomy, to examine the microanatomy of the cornea in three dimensions and in large tissue volumes. The results show that corneal keratocytes occupy a significantly greater tissue volume than was previously thought, and there is a clear orthogonality in cell and matrix organization, quantifiable by Fourier analysis. Three-dimensional reconstructions reveal actin-associated tubular cell protrusions, reminiscent of filopodia, but extending more than 30 µm into the extracellular space. The highly extended network of these membrane-bound structures mirrors the alignment of collagen bundles and emergent lamellae and, we propose, plays a fundamental role in dictating the orientation of collagen in the developing cornea.

Large proteoglycan complexes and disturbed collagen architecture in the corneal extracellular matrix of mucopolysaccharidosis type VII (Sly syndrome).

PURPOSE Deficiencies in enzymes involved in proteoglycan (PG) turnover underlie a number of rare mucopolysaccharidoses (MPS), investigations of which can considerably aid understanding of the roles of PGs in corneal matrix biology. Here, the authors analyze novel pathologic changes in MPS VII (Sly syndrome) to determine the nature of PG-collagen associations in stromal ultrastructure. METHODS Transmission electron microscopy and electron tomography were used to investigate PG-collagen architectures and interactions in a cornea obtained at keratoplasty from a 22-year-old man with MPS VII, which was caused by a compound heterozygous mutation in the GUSB gene. RESULTS Transmission electron microscopy showed atypical morphology of the epithelial basement membrane and Bowmans layer in MPS VII. Keratocytes were packed with cytoplasmic vacuoles containing abnormal glycosaminoglycan (GAG) material, and collagen fibrils were thinner than in normal cornea and varied considerably throughout anterior (14-32 nm), mid (13-42 nm), and posterior (17-39 nm) regions of the MPS VII stroma. PGs viewed in three dimensions were striking in appearance in that they were significantly larger than PGs in normal cornea and formed highly extended linkages with multiple collagen fibrils. CONCLUSIONS Cellular changes in the MPS VII cornea resemble those in other MPS. However, the wide range of collagen fibril diameters throughout the stroma and the extensive matrix presence of supranormal-sized PG structures appear to be unique features of this disorder. The findings suggest that the accumulation of stromal chondroitin-, dermatan-, and heparan-sulfate glycosaminoglycans in the absence of β-glucuronidase-mediated degradation can modulate collagen fibrillogenesis.

Human junctophilin-2 undergoes a structural rearrangement upon binding PtdIns(3,4,5)P3 and the S101R mutation identified in hypertrophic cardiomyopathy obviates this response

JP2 (junctophilin-2) is believed to hold the transverse tubular and jSR (junctional sarcoplasmic reticulum) membranes in a precise geometry that facilitates excitation–contraction coupling in cardiomyocytes. We have expressed and purified human JP2 and shown using electron microscopy that the protein forms elongated structures ~15 nm long and 2 nm wide. Employing lipid-binding assays and quartz crystal microbalance with dissipation we have determined that JP2 is selective for PS (phosphatidylserine), with a Kd value of ~0.5 μM, with the N-terminal domain mediating this interaction. JP2 also binds PtdIns(3,4,5)P3 at a different site than PS, resulting in the protein adopting a more flexible conformation; this interaction is modulated by both Ca2+ and Mg2+ ions. We show that the S101R mutation identified in patients with hypertrophic cardiomyopathy leads to modification of the protein secondary structure, forming a more flexible molecule with an increased affinity for PS, but does not undergo a structural transition in response to binding PtdIns(3,4,5)P3. In conclusion, the present study provides new insights into the structural and lipid-binding properties of JP2 and how the S101R mutation may have an effect upon the stability of the dyad organization with the potential to alter JP2–protein interactions regulating Ca2+ cycling.

Electron tomography reveals multiple self-association of chondroitin sulphate/dermatan sulphate proteoglycans in Chst5-null mouse corneas.

The spatial distribution of collagen fibrils in the corneal stroma is essential for corneal transparency and is primarily regulated by extrafibrillar proteoglycans, which are multi-functional polymers that interact with hybrid type I/V collagen fibrils. In order to understand more about proteoglycan organisation and collagen associations in the cornea, three-dimensional electron microscopy reconstructions of collagen-proteoglycan interactions in the anterior, mid and posterior stroma from a Chst5 knockout mouse, which lacks a keratan sulphate sulphotransferase, were obtained. Both longitudinal and transverse section show sinuous, oversized proteoglycans with near-periodic, orthogonal off-shoots. In many cases, these proteoglycans traverse over 400nm of interfibrillar space interconnecting over 10 collagen fibrils. The reconstructions suggest that multiple chondroitin sulphate/dermatan sulphate proteoglycans have aggregated laterally and, possibly, end-to-end, with orthogonal extensions protruding from the main electron-dense stained filament. We suggest possible mechanisms as to how sulphation differences may lead to this increase in aggregation of proteoglycans in the Chst5-null mouse corneal stroma and how this relates to proteoglycan packing in healthy corneas.

3-D Reconstruction of the Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking T-Tubules: This Organization is Perturbed in Heart Failure

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

A computational model of spatio-temporal cardiac intracellular calcium handling with realistic structure and spatial flux distribution from sarcoplasmic reticulum and t-tubule reconstructions

Intracellular calcium cycling is a vital component of cardiac excitation-contraction coupling. The key structures responsible for controlling calcium dynamics are the cell membrane (comprising the surface sarcolemma and transverse-tubules), the intracellular calcium store (the sarcoplasmic reticulum), and the co-localisation of these two structures to form dyads within which calcium-induced-calcium-release occurs. The organisation of these structures tightly controls intracellular calcium dynamics. In this study, we present a computational model of intracellular calcium cycling in three-dimensions (3-D), which incorporates high resolution reconstructions of these key regulatory structures, attained through imaging of tissue taken from the sheep left ventricle using serial block face scanning electron microscopy. An approach was developed to model the sarcoplasmic reticulum structure at the whole-cell scale, by reducing its full 3-D structure to a 3-D network of one-dimensional strands. The model reproduces intracellular calcium dynamics during control pacing and reveals the high-resolution 3-D spatial structure of calcium gradients and intracellular fluxes in both the cytoplasm and sarcoplasmic reticulum. We also demonstrated the capability of the model to reproduce potentially pro-arrhythmic dynamics under perturbed conditions, pertaining to calcium-transient alternans and spontaneous release events. Comparison with idealised cell models emphasised the importance of structure in determining calcium gradients and controlling the spatial dynamics associated with calcium-transient alternans, wherein the probabilistic nature of dyad activation and recruitment was constrained. The model was further used to highlight the criticality in calcium spark propagation in relation to inter-dyad distances. The model presented provides a powerful tool for future investigation of structure-function relationships underlying physiological and pathophysiological intracellular calcium handling phenomena at the whole-cell. The approach allows for the first time direct integration of high-resolution images of 3-D intracellular structures with models of calcium cycling, presenting the possibility to directly assess the functional impact of structural remodelling at the cellular scale.