Biology subjects

Calve, S.

Publications and source records attributed to Calve, S..

12 recordsLinked to original sources

bioRxiv2026

Quantifying the role of hyaluronic acid on collagen fiber mechanics via femtosecond laser ablation

Despite therapeutic use of hyaluronic acid (HA) for tendon injuries and tendinopathies, the role of HA in tendon mechanical behavior remains poorly understood. Here, we describe the development of a method that integrates femtosecond laser ablation, second harmonic generation imaging, and mechanical testing to quantify collagen fiber recoil within intact murine tendons under load. Fiber recoil following ablation is used as a measure of collagen fiber sliding and local viscoelastic behavior. Aging and enzymatic depletion of endogenous HA both significantly reduce tendon HA content and decrease elastic, viscoelastic, and total collagen fiber recoil, indicating diminished fiber sliding. To restore HA, low molecular weight methacrylated HA (MeHA) is diffused into tendons and retained through photocrosslinking. MeHA supplementation recovers collagen fiber recoil in HA-depleted and aged tendons, restoring micromechanical behavior toward adult control levels. Quasi-linear viscoelastic modeling further reveals HA-dependent changes in relaxation behavior that are not evident from conventional macroscale mechanical measurements alone. These findings identify femtosecond laser ablation as a powerful platform to quantify fiber-scale mechanics and investigate extracellular matrix contributions to tissue material properties, the results of which specifically isolate HA as an important contributor to tendon collagen fiber sliding and mechanical properties.

bioengineering↗

bioRxiv2025

Fiber Type and Stimulus Determine Progression of Skeletal Muscle Atrophy

BackgroundSkeletal muscle atrophy is prevalent worldwide and is a major detractor from length and quality of life. It is often diagnosed and treated as a single disorder, but the causal stimuli and progression of atrophy vary widely. Malnutrition and disuse are two common causes of muscle atrophy, and despite their prevalence and extensive characterization, there have been no direct comparisons of how these two types of atrophy progress and whether they differentially affect skeletal muscle fiber types. The purpose of this study is to directly compare atrophy from fasting and disuse and provide a transcriptomic resource for future research on both conditions. MethodsWe fasted or hindlimb suspended (HS) two cohorts of 12-week-old female C57/bl6 mice. Mice were fasted for up to 72 hours to induce malnutrition atrophy or were hindlimb suspended for 0, 3, 7, 14, or 28 days to induce disuse atrophy. At each timepoint, mice were euthanized and three muscles (tibialis anterior (TA), extensor digitorum longus (EDL), and soleus) were weighed and collected for RNA sequencing. Atrophy progression and gene expression changes were compared across muscle fiber types and atrophy stimuli. ResultsWe found differences in atrophy progression between muscle fiber types based on fiber twitch speed and atrophy stimulus. Fasted mice lost 25% of their body weight and 23% of fast-twitch TA mass with little change in soleus. In contrast, HS mice lost 40% of the slower-twitch soleus but the effect on the TA was negligible. Gene expression varied in response to both atrophy stimuli, but a greater number of genes changed with fasting compared to HS in the EDL and soleus. By muscle type, a greater transcriptional shift occurred in the EDL with fasting while the soleus showed more gene changes during HS. Enrichment analysis of transcriptional changes showed similarities (downregulation in muscle growth pathways) and differences (increased fatty acid metabolism in fasting and increased neuronal activity in HS) between atrophy stimuli. ConclusionsAtrophy progression varies based on stimuli and muscle fiber type. This study provides a large, matched data set where the effects of different atrophic stimuli can be easily and directly compared in multiple fiber types. To our knowledge, this is the first study to closely compare these two atrophy stimuli in a muscle type-specific context. This work demonstrates that atrophy is not a single disorder and that the development of therapies may need to be tailored to the atrophic stimulus.

molecular biology↗

bioRxiv2022

Multiscale Mechanical Characterization and Computational Modeling of Fibrin Gels

Fibrin is a naturally occurring protein network that forms a temporary structure to enable remodeling during wound healing. It is also a common tissue engineering scaffold because the structural properties can be controlled. However, to fully characterize the wound healing process and improve the design of regenerative scaffolds, understanding fibrin mechanics at multiple scales is necessary. Here, we present a strategy to quantify both the macroscale (1 - 10 mm) stress-strain response and the deformation of the mesoscale (10 - 1000 m) network structure during unidirectional tensile tests. The experimental data is then used to inform a computational model to accurately capture the mechanical response of fibrin gels. Simultaneous mechanical testing and confocal microscopy imaging of fluorophore-conjugated fibrin gels revealed up to an 88% decrease in volume coupled with increase in volume fraction in deformed gels, and non-affine fiber alignment in the direction of deformation. Combination of the computational model with finite element analysis enabled us to predict the strain fields that were observed experimentally within heterogenous fibrin gels with spatial variations in material properties. These strategies can be expanded to characterize and predict the macroscale mechanics and mesoscale network organization of other heterogeneous biological tissues and matrices. Statement of SignificanceFibrin is a naturally-occurring scaffold that supports cellular growth and assembly of de novo tissue and has tunable material properties. Characterization of meso- and macro-scale mechanics of fibrin gel networks can advance understanding of the wound healing process and impact future tissue engineering approaches. Using structural and mechanical characteristics of fibrin gels, a theoretical and computational model that can predict multiscale fibrin network mechanics was developed. These data and model can be used to design gels with tunable properties.

bioengineering↗

bioRxiv2022

Mechanical loading is required for initiation of extracellular matrix deposition at the developing murine myotendinous junction

The myotendinous junction (MTJ) contributes to the generation of motion by connecting muscle to tendon. At the adult MTJ, a specialized extracellular matrix (ECM) is thought to contribute to the mechanical integrity of the muscle-tendon interface, but the factors that influence MTJ formation during mammalian development are unclear. Here, we combined 3D imaging and proteomics with murine models in which muscle contractility and patterning are disrupted to resolve morphological and compositional changes in the ECM during MTJ development. We found that MTJ-specific ECM deposition can be initiated via static loading due to growth; however, it required cyclic loading to develop a mature morphology. Furthermore, the MTJ can mature without the tendon terminating into cartilage. Based on these results, we describe a model wherein MTJ development depends on mechanical loading but not insertion into an enthesis.

developmental biology↗

bioRxiv2022

Extracellular matrix deposition precedes muscle-tendon integration during murine forelimb morphogenesis

The development of a functional vertebrate musculoskeletal system requires the combination of contractile muscle and extracellular matrix (ECM)-rich tendons that transmit muscle-generated force to bone. Despite the different embryologic origins, muscle and tendon integrate at the myotendinous junction (MTJ) to seamlessly connect cells and ECM across this interface. While the cell-cell signaling factors that direct development have received considerable attention, how and when the ECM linking these tissues is deposited remains unknown. To address this gap, we analyzed the 3D distribution of different ECM and the influence of skeletal muscle in forelimbs from wildype (WT) and muscle-less Pax3Cre/Cre mice. At E11.5, prior to MTJ integration, an aligned ECM was present at the presumptive insertion of the long triceps into the WT ulna. Mechanically robust tendon-like and muscle compartmentalization structures, positive for type I collagen, type V collagen, and fibrillin-2, still formed when muscle was knocked out. However, MTJ-specific ECM was not observed when muscle was absent. Our results show that an ECM-based template forms independent of muscle, but muscle is needed for the proper assembly of ECM at the MTJ. Summary statementAn aligned ECM template connects tendon and muscle during limb development, independent of muscle progenitor migration into the limb; however, the assembly of MTJ-specific ECM requires the presence of muscle.

developmental biology↗

bioRxiv2021

AN INTEGRATIVE BIOLOGY APPROACH TO QUANTIFY THE BIODISTRIBUTION OF AZIDOHOMOALANINE IN VIVO

Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. Since its conception, ncAA labeling has been applied to study many in vitro systems and more recently the in vivo proteomes of complex organisms such as rodents. In vivo labeling is typically achieved by introducing ncAAs into diet, which requires extended labeling times. We have previously demonstrated that labeling the murine proteome is feasible via injection of azidohomoalanine (Aha), a ncAA and methionine (Met) analog, without the need for Met depletion. With the ability to isolate NSPs without applying stress from dietary changes, Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues. Furthermore, studies of physiological effects of ncAA administration have been limited to gross observation of animal appearance. To address these gaps, we created a deterministic, compartmental model of the biokinetic transport and incorporation of Aha in mice. Parameters were informed from literature and experimentally. Model results demonstrate the ability to predict Aha distribution and labeling under a variety of dosing paradigms and confirms the use of the model as a tool for design of future studies. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing the plasma metabolome following Aha injection. We show that Aha administration does not significantly perturb cellular functions as reflected by an unchanged plasma metabolome compared to non-injected controls. Author SummaryAs the machinery of life, proteins play a key role in dynamic processes within an organism. As such, the response of the proteome to perturbation is increasingly becoming a critical component of biological and medical studies. Dysregulation of protein mechanisms following exposure to experimental treatment conditions can implicate physiological mechanisms of health and disease, elucidate toxin/drug response, and highlight potential targets for novel therapies. Traditionally, these questions have been probed by studying perturbations in total proteins following an experimental treatment. However, the proteome is expansive and noisy, often an early response can be indiscernible against the background of unperturbed proteins. Here, we apply a technique to selectively label newly synthesized proteins, which enables capturing early changes in protein behavior. We utilize an amino acid analog that naturally incorporates into proteins, and investigate the tissue distribution, protein labeling efficiency, and potential physiological impact of this analog in mice. Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We further present a computational model that can be used to guide future experiments utilizing this technique to study proteomic responses to stimuli.

bioengineering↗

bioRxiv2020

Nuclear stiffness decreases with disruption of the extracellular matrix in living tissues

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. The extracellular environment is physically linked to the nuclear envelope and provides cues to maintain nuclear structure and cellular homeostasis regulated in part by mechanotransduction mechanisms. However, little is known about how structure and properties of the extracellular matrix in living tissues impacts nuclear mechanics, and current experimental challenges limit the ability to detect and directly measure nuclear mechanics while cells are within the native tissue environment. Here, we hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. We aimed to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, we expanded an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue. We validated this technique by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, we found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, we demonstrated the capability for cell and nucleus elastography using the AFM needle-tip technique. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

biophysics↗

bioRxiv2020

3D mapping reveals a complex and transient interstitial matrix during murine renal development

BackgroundThe extracellular matrix (ECM) is a network of proteins and glycosaminoglycans that provides structural and biochemical cues to cells. In the kidney, the ECM is critical for nephrogenesis; however, the dynamics of ECM composition and how it relates to 3D structure during development is unknown. MethodsUsing embryonic day (E)14.5, E18.5, postnatal day (P)3, and adult kidneys, we fractionated proteins based on differential solubilities, performed liquid chromatography tandem-mass spectrometry, and identified changes in ECM protein content (matrisome). Decellularized kidneys were stained for ECM proteins and imaged in 3D using confocal microscopy. ResultsWe observed an increase in interstitial ECM that connect the stromal mesenchyme to the basement membrane (TNXB, COL6A1, COL6A2, COL6A3) between the embryo and adult, and a transient elevation of interstitial matrix proteins (COL5A2, COL12A1, COL26A1, ELN, EMID1, FBN1, LTBP4, THSD4) at perinatal timepoints. Basement membrane proteins critical for metanephric induction (FRAS1, FREM2) were highest in the embryo, whereas proteins necessary for glomerular basement membrane integrity (COL4A3, COL4A4, COL4A5, LAMB2) were more abundant in the adult. 3D visualization revealed a complex interstitial matrix that dramatically changed over development, including the perinatal formation of fibrillar structures that appear to support the medullary rays. ConclusionBy correlating 3D ECM spatiotemporal organization with global protein abundance, we identified novel changes in the interstitial matrix during kidney development. This new information regarding the ECM in developing kidneys offers the potential to inform the design of regenerative scaffolds that can guide nephrogenesis in vitro. Significance statementEnd-stage renal disease is increasing and there are a limited number of organs available for transplantation. Therefore, researchers have focused on understanding how cellular signaling influences kidney development to expand strategies to rebuild a kidney. However, the extracellular matrix (ECM), another critical component that biomechanically regulates nephrogenesis, has been largely neglected. This paper combines proteomics and 3D imaging of the murine kidney to resolve previously undescribed dynamics of the interstitial matrix in the cortex and corticomedullary junction during development. Combined with cell and growth factors, scaffolds modeled after the composition and organization of the developmental ECM have the potential to improve tissue engineering models of the kidney, like organoids.

developmental biology↗

bioRxiv2020

Percolation of Microparticle Matrix Promotes Cell Migration and Integration while Supporting Native Tissue Architecture

Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, we developed a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture. We utilized hyaluronic acid-based hydrogels, amorphously packed with acellular articular cartilage tissue particulated to ~125-250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300kPa. Remarkably, primary chondrocytes recellularized particles within 48 hours, a process driven by chemotaxis, exhibited distributed cellularity in large engineered composites, and expressed genes consistent with native cartilage repair. We additionally demonstrated broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in a subcutaneous in vivo mouse model. Our findings suggest optimal strategies and material architectures to balance concurrent demands for large-scale mechanical properties while also supporting integration of dense musculoskeletal and connective tissues.

bioengineering↗

bioRxiv2020

Extracellular matrix protein composition dynamically changes during murine forelimb development

The extracellular matrix (ECM) is an integral part of multicellular organisms, connecting different cell layers and tissue types. During morphogenesis and growth, tissues undergo substantial reorganization involving cellular proliferation, migration, and differentiation. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development. We utilized tissue fractionation and mass spectrometry to define ECM protein (matrisome) dynamics during murine forelimb development and resolved significant differences in ECM composition as a function of development, disease and tissue type. Additionally, we used bioorthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized ECM within the developing forelimb. We demonstrate the feasibility of using BONCAT to enrich for newly synthesized matrisome components and identified differences in ECM synthesis between morphogenesis and growth. This resource will guide future research investigating the role of the matrisome during complex tissue development.

developmental biology↗

bioRxiv2019

Three dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval

Single-molecule localization microscopy is a powerful tool in visualizing organelle structures, interactions, and protein functions in biological research. However, whole-cell and tissue specimens challenge the achievable resolution and depth of nanoscopy methods. As imaging depth increases, photons emitted by fluorescent probes, the sole source of molecular positions, were scattered and aberrated, resulting in image artifacts and rapidly deteriorating resolution. We propose a method to allow constructing the in situ 3D response of single emitters directly from single-molecule dataset and therefore allow pin-pointing single-molecule locations with limit-achieving precision and uncompromised fidelity through whole cells and tissues. This advancement expands the routine applicability of super-resolution imaging from selected cellular targets near coverslips to intra- and extra-cellular targets deep inside tissues. We demonstrate this across a range of cellular-tissue architectures from mitochondrial networks, microtubules, and nuclear pores in 2D and 3D cultures, amyloid-{beta} plaques in mouse brains to developing cartilage in mouse forelimbs.

biophysics↗

bioRxiv2018

The Nucleus Mediates Mechanosensitive Reorganization of Epigenetically Marked Chromatin During Cardiac Maturation and Pathology

Environmental mechanical cues are critical to guide cell fate. Forces transmit to the nucleus through the Linker of Nucleo- and Cytoskeleton (LINC) complex and are thought to influence the organization of chromatin that is related to cell differentiation; however, the underlying mechanisms are unclear. Here, we investigated chromatin reorganization during murine cardiac development and found that cardiomyocytes establish a distinct architecture characterized by relocation of H3K9me3-modified chromatin from the nuclear interior to the periphery and co-localization to myofibrils. This effect was abrogated in stiff environments that inhibited cardiomyocyte contractility, or after LINC complex disruption, and resulted in the relocation of H3K27me3-modified chromatin instead. By generating high-resolution intra-nuclear strain maps during cardiomyocyte contraction, we discovered that the reorganization of H3K9me3-marked chromatin is influenced by tensile, but not compressive, nuclear strains. Our findings highlight a new role for nuclear mechanosensation in guiding cell fate through chromatin reorganization in response to environmental cues.

cell biology↗