Biology subjects

Rutten, L.

Publications and source records attributed to Rutten, L..

13 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

Vaccine Elicitation of HIV-1 Neutralizing Antibodies Against Both V2 Apex and Fusion Peptide in Rhesus Macaques

Broadly neutralizing antibodies targeting multiple sites of HIV-1 Env vulnerability can be induced by infection, but simultaneous elicitation of neutralizing antibodies (NAbs) against multiple epitopes has not yet been achieved by vaccination. In this study, we designed a dual-epitope vaccine targeting both fusion peptide (FP) and V2 apex and evaluated its capacity to induce NAbs against both epitopes in rhesus macaques. This vaccine combined an FP conjugate with a cocktail of engineered Env trimers with enhanced V2 apex recognition and increased antigen retention in lymph nodes. Immunization of macaques with the dual-epitope vaccine elicited >1000-fold higher autologous tier 2-neutralization titers than the wildtype Env trimer and enhanced heterologous NAb breadth. Both FP and V2-apex monoclonal antibodies (mAb) were isolated from immunized macaques and showed heterologous neutralization with genetic and structural signatures that were similar to well-characterized FP and V2 apex bNAbs, although the V2 apex mAbs showed incomplete maturation. These results demonstrate proof-of-concept for simultaneous vaccine elicitation of NAbs against multiple sites of Env vulnerability, which will likely be critical for an effective HIV-1 vaccine. HIGHLIGHTSO_LIDesigned a dual-epitope vaccine targeting both fusion peptide (FP) and V2 apex C_LIO_LIV2-SET Env trimer conferred higher binding of V2 apex bNAbs, longer retention in draining lymph nodes, and >1000-fold higher induction of autologous neutralization titers compared with wildtype Env trimer C_LIO_LIDual-epitope vaccine enhanced serum tier 2 neutralization breadth C_LIO_LIDual-epitope vaccine elicited FP- and V2 apex-specific neutralizing mAbs with genetic signatures similar to well-characterized FP and V2 apex bNAbs C_LI

immunology↗

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↗

bioRxiv2023

Unravelling the relationship between isolated bone matrix vesicles and forming mineral at the nanometer scale

Matrix vesicles (MVs) are involved in the initial deposition of hydroxyapatite (HAp) during bone mineralization, but their mechanism of action is not yet fully understood. In vitro studies propose two pathways by which MVs can trigger HAp precipitation: the first is mediated by their enhanced phosphatase activity, and the second suggested to depend on structural components present in MVs to mediate nucleation directly from soluble ions without the requirement of phosphatase activity. However, the relevance of these two pathways for bone mineralization and the relationship between MVs and forming mineral in such in vitro experiments remains unclear. Here, we used near-native cryoTEM nanoscale imaging in combination with bulk characterizations to disentangle the content and action of MVs during in vitro mineralization. We show that MVs isolation by conventional ultracentrifugation results in heterogeneous dispersions containing non-vesicular particles, including collagens and proteoglycans, in addition to bilayered vesicles. The separation of phosphatase-enriched MVs from non-vesicular particles and comparative mineralization experiments demonstrated that the ability of MVs to induce fast mineralization, independently of phosphatase activity, depends on the presence of non-vesicular particles. Therefore, we conclude that the primary pathway by which MVs trigger mineralization is through the action of their resident phosphatase enzymes, with the direct mineral nucleation to be a secondary event consequential of their membrane components. Lastly, we observed mineral formation restricted to the extravesicular space or in close proximity to the membrane interface, suggesting that the relationship between MVs and forming mineral is more intricate than previously understood.

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↗

bioRxiv2022

Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.

cell biology↗

bioRxiv2022

Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike

The SARS-CoV-2 spike protein is the target of neutralizing antibodies and the immunogen used in all currently approved vaccines. The global spread of the virus has resulted in emergence of lineages which are of concern for the effectiveness of immunotherapies and vaccines based on the early Wuhan isolate. Here we describe two SARS-CoV-2 isolates with large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis showed that the deletions result in complete reshaping of the antigenic surface of the NTD supersite. The remodeling of the NTD affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite for both spike variants. A unique escape mechanism with high antigenic impact observed in the {Delta}N135 variant was based on the loss of the Cys15-Cys136 disulfide due to the P9L-mediated shift of the signal peptide cleavage site and deletion of residues 136-144. Although the observed large loop and disulfide deletions are rare, similar modifications became independently established in several other lineages, highlighting the possibility of a general escape mechanism via the NTD supersite. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.

molecular biology↗

bioRxiv2021

Ad26.COV2.S-elicited immunity protects against G614 spike variant SARS-CoV-2 infection in Syrian hamsters and does not enhance respiratory disease in challenged animals with breakthrough infection after sub-optimal vaccine dosing

Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1x109 vp and 1x1010 vp elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers which was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these pre-clinical data confirm efficacy of a 1-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.

immunology↗

bioRxiv2020

Ad26-vector based COVID-19 vaccine encoding a prefusion stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses

Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g. prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S1) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild type signal peptide was best suited for the correct cleavage needed for a natively-folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-{gamma}. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

immunology↗

bioRxiv2017

Parallel loss of symbiosis genes in relatives of nitrogen-fixing non-legume Parasponia

Rhizobium nitrogen-fixing nodules are a well-known trait of legumes, but nodules also occur in other plant lineages either with rhizobium or the actinomycete Frankia as microsymbiont. The widely accepted hypothesis is that nodulation evolved independently multiple times, with only a few losses. However, insight in the evolutionary trajectory of nodulation is lacking. We conducted comparative studies using Parasponia (Cannabaceae), the only non-legume able to establish nitrogen fixing nodules with rhizobium. This revealed that Parasponia and legumes utilize a large set of orthologous symbiosis genes. Comparing genomes of Parasponia and its non-nodulating relative Trema did not reveal specific gene duplications that could explain a recent gain of nodulation in Parasponia. Rather, Trema and other non-nodulating species in the Order Rosales show evidence of pseudogenization or loss of key symbiosis genes. This demonstrates that these species have lost the potential to nodulate. This finding challenges a long-standing hypothesis on evolution of nitrogen-fixing symbioses, and has profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

plant biology↗