Biology subjects

Macias-Sanchez, E.

Publications and source records attributed to Macias-Sanchez, E..

9 recordsLinked to original sources

bioRxiv2026

Uncompromised, multimodal, multiscale structural analysis of the hierarchically organization in mineralized tissues

We present a live-to-cryo correlative imaging workflow for multiscale structural and chemical analysis of biological tissues in their near-native state. The method integrates live super-resolution fluorescence microscopy, live and cryogenic Raman spectroscopy, and targeted cryogenic focused ion beam/scanning electron microscopy, transmission electron microscopy, electron tomography, energy dispersive X-ray spectroscopy, and electron diffraction. This approach enables precise 3D targeting and nanoscale imaging of selected regions across four orders of magnitude in spatial resolution, while preserving ultrastructure and chemical composition. Using regenerating zebrafish scales as a benchmark, we visualize collagen fibril orientation, local matrix density, and mineral composition within the extracellular matrix. We identify a plywood-like architecture of unmineralized collagen with orientation-independent density variation, and reveal curved, acidic phosphate-rich mineral platelets aligned with collagen fibrils. This workflow establishes a generalizable strategy for comprehensive 3D correlative analysis of hybrid tissues, and opens new opportunities for studying native structure-function relationships at the interface of biology and materials science.

biophysics↗

bioRxiv2025

Overglycosylation introduces local changes in triple helix alignment in collagen type I fibril structure

Collagen fibrils constitute the structural scaffold of bone, and their hierarchical organization is central to biomineralization. While hydroxylation and glycosylation of lysine residues are well-known collagen post-translational modifications, their structural consequences remain poorly understood. Here we show that excess of glycosylation of hydroxylysine residues leads to significant alterations in the local packing of collagen molecules within the fibrils. By prolonging the enzymatic modification window during helix folding through the use of cyclosporin A, an increase in double glycosylation was observed at specific sites. These overglycosylated residues were pinpointed by mass spectrometry, while cryogenic TEM revealed fibrils with reduced diameters and distinct displacements of defined sub-bands within the D-period, without altering the overall periodicity. By mapping the modified residues onto the quarter-staggered model, we have been able to correlate site-specific glycosylation with sub-band shifts, linking chemical modification to supramolecular order. These results provide molecular-level evidence that collagen glycosylation is an active determinant of fibril structure. Such insights not only advance fundamental understanding of collagen assembly but could also illuminate mechanisms underlying bone fragility disorders, including osteogenesis imperfecta, that feature altered glycosylation.

biochemistry↗

bioRxiv2025

Combined Raman Microscopy and Transmission Electron Microscopy shows the co-existence of whitlockite crystals and carbonated hydroxyapatite-mineralized collagen fibrils in human calcified aortic valves

Aortic valve stenosis (AS), the leading valvular disease in aging populations, is driven by complex extracellular matrix (ECM) modifications and progressive calcification. Despite its clinical significance, the molecular mechanisms underlying AS remain poorly understood, limiting therapeutic options to invasive valve replacement. This study employs Raman microscopy, a label-free imaging technique, and its integration with transmission electron microscopy (TEM) to elucidate the biochemical and ultrastructural changes in stenotic aortic valves. High-resolution spectroscopic imaging revealed distinct extracellular matrix modification across different regions, involving elastin degradation, cholesterol deposition, and mineral formation, comprising not only hydroxyapatite (cHAp) but also whitlockite. Elastin-rich domains associated with cHAp deposition exhibited cross-link degradation, while collagen matrices supported mineralized phases with varying mineral-to-matrix ratios that, in heavily mineralized regions, went far above those of mature human bone. For the first time we demonstrated whitlockite as a mineral deposit in calcified aortic valves, in areas with different degrees of calcification. This implies that this Mg containing mineral, which has been considered a precursor to cHAp in pathological calcification, forms independently, challenging prevailing models of calcification. The combination of Raman and TEM showed how bone-like cHAp mineralized collagen matrix in later stages engulfs the initial non-physiological whitlockite deposits. This correlative multimodal approach advances our understanding of AS by capturing spatially resolved chemical and structural dynamics at the nanoscale. The findings highlight Raman microscopys potential for probing calcification mechanisms across diverse tissue types and suggest its role in identifying novel therapeutic targets. This study underscores the value of integrative imaging methodologies in unraveling complex pathological processes and advancing patient care.

biochemistry↗

bioRxiv2025

Two Transport Routes for Fetuin-A-based Mineral Complexes in Osteogenic Matrix Mineralization

Living organisms must transport calcium and phosphate at high concentrations to enable bone formation without triggering uncontrolled mineral precipitation. Fetuin-A binds calcium phosphate to form soluble calciprotein complexes, but how these complexes contribute to physiological mineralization has remained unclear. Here we show that Fetuin-A-based mineral complexes exist in functionally distinct states that determine mineral bioavailability. Using cryogenic and liquid-phase electron microscopy, biochemical analysis, and osteoblast cell cultures, we demonstrate that small, chemically labile calciprotein monomers directly mineralize collagen fibrils, whereas larger, chemically matured primary calciprotein particles cannot. Instead, these particles require cellular uptake and lysosomal processing to release mineral for matrix deposition. This functional divergence arises from a irreversible chemical transformation of the mineral phase that drives the primary CPP assembly. Together, our findings establish nanoscale chemical maturation as a key control point that separates mineral transport from mineralization, reframing our understanding both physiological bone formation and pathological calcification.

biochemistry↗

bioRxiv2024

Organic macromolecules bind a significant proportion of the precursor calcium for nacre formation.

The mechanism of nacre formation in gastropods involves a vesicular system that transports organic and mineral precursors from the mantle epithelium to the mineralization chamber. Between them lies the surface membrane, a thick organic structure that covers the mineralization chamber and the forming nacre. The surface membrane is a dynamic structure that grows by the addition of vesicles on the outer side and recedes by the formation of interlamellar membranes on the inner side. By using a combination of electron microscopy imaging and spectroscopy, we have monitored the journey of the vesicles from the mantle epithelium to the mineralization chamber, focusing on the elemental composition of the organic structures at each stage. Our data reveal that transport occurs in lipid bilayer vesicles through exocytosis from the outer mantle epithelium. After release into the surface membrane, chitin undergoes a process of self-assembly and interaction with proteins, resulting in progressive changes of the internal structure of the surface membrane until the final structure of the interlamellar membranes is acquired. Finally, these detach from the inner side of the surface membrane. Elemental analysis revealed the transport of a considerable amount of calcium bound to proteins, likely forming calcium-protein complexes.

biophysics↗

bioRxiv2023

A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells

Liquid phase electron microscopy (LP-EM) has emerged as a powerful technique for in-situ observation of material formation in liquid. However, monitoring these processes requires the repeated interaction of the electron beam with the aqueous environment leading to the decomposition of water molecules (radiolysis), which affects the formation processes under investigation. Graphenes ultra-conductive properties have made it an efficient way to mitigate this problem, as it acts as an electron scavenger when used as a window material. Using the strategy, the process of interest is initiated when the graphene liquid cells (GLCs) are sealed. This means that the process cannot be imaged at early time points since microscope preparation and initiation of image acquisition at the region of interest are time-consuming. Here we report a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses the most significant limitations of the graphene liquid cells, while combining the advantages of fluorescence and electron microscopy. This workflow allows imaging to be initiated at a predetermined space and time by vitrifying and thawing at a selected time point. We demonstrate the workflow first by observing multiple day crystallization processes and highlight its potential by observing a biological process: the complexation of calciprotein particles. With this observation, we show the exciting possibilities for LP-EM in biology.

biochemistry↗

bioRxiv2022

A 3D CELL-FREE BONE MODEL SHOWS COLLAGEN MINERALIZATION IS A PHYSICOCHEMICAL PROCESS DRIVEN AND CONTROLLED BY THE MATRIX

Osteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization and different degrees of mineralization where the intrafibrillar and extrafibrillar mineral is intergrown into one continuous network of oriented crystals. While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization, and suggest that this process could be simply driven by the interactions of the mineral with extracellular matrix. Through the remineralization of demineralized bone matrix, we demonstrate the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement. We then explore this cell-free in vitro system as a realistic, functional model for the in situ investigation of matrix-controlled mineralization processes. Combined Raman and electron microscopy indicates that glycosaminoglycans play a more prominent role than generally assumed in the matrix-mineral interactions. Our experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral.

biochemistry↗

bioRxiv2019

Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases

Complex hierarchical structure governs emergent properties in soft biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multiscale structure and composition of the mussel byssus cuticle before, during and after formation to gain further insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticles function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are separated into catechol-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with Fe contained in the sulfur-rich matrix and V being relegated to the granules, coordinated by catechol. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic protein components within secretory vesicles, resulting in a meso-scale structuring, critical to the cuticles advanced function.

biophysics↗

bioRxiv2019

Three-dimensional Structural Interrelations between Cells, Extracellular Matrix and Mineral in Vertebrate Mineralization

The spatial-temporal relationship between cells, extracellular matrices and mineral deposits is fundamental for an improved understanding mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of microns in range. These parameters are necessary to yield fine ultrastructural details while encompassing tissue domains sufficient to provide a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a novel complex cellular network in highly mineralized tendon aspects, where [~]100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of [~]40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich globules appear between the fibrils and, with time, mineral propagates along and within collagen. These close associations between tenocytes, canaliculi, small channels, collagen and mineral suggest a new concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.\n\nSignificance StatementThe basic mechanism by which vertebrate collagenous tissues are mineralized is still not fully elucidated, despite the importance of this process for skeletal formation and regeneration. Through three-dimensional imaging of the cellular network together with the extracellular matrix and mineral deposits, the present work investigates normally mineralizing avian leg tendon as a model system for vertebrates in general. The data support a mechanism where mineral ions and possible mineral precursors are initially present in interfibrillar collagen spaces and are subsequently translocated to neighboring collagen fibrils. Mineral particles then nucleate in association with collagen to form the well known collagen-mineral composite material of the skeleton.

bioengineering↗