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1.
Biophys J ; 2022 Nov 29.
Article in English | MEDLINE | ID: mdl-36451545

ABSTRACT

Cells translate mechanical cues from the extracellular matrix (ECM) into signaling that can affect the nucleus. One pathway by which such nuclear mechanotransduction occurs is a signaling axis that begins with integrin-ECM bonds and continues through a cascade of chemical reactions and structural changes that lead to nuclear translocation of the YAP/TAZ which is independent of hippo pathway. This signaling axis is self-reinforcing, with stiff ECM promoting integrin binding and thus facilitating polymerization and tension in the cytoskeletal contractile apparatus, which can compress nuclei, open nuclear pore channels, and enhance nuclear accumulation of YAP/TAZ. We previously developed a computational model of this mechanosensing axis for the linear elastic ECM by assuming that there is a linear relationship between the nucleocytoplasmic ratio of YAP/TAZ and nuclear flattening. Here, we extended our previous model to more general ECM behaviors (e.g., viscosity, viscoelasticity and viscoplasticity) and included more details about YAP/TAZ translocation dynamics based on nuclear deformation. This model was predictive of diverse mechanosensing responses in a broad range of cells. Results support the hypothesis that this diverse range of mechanosensing phenomena across many cell types arises from a simple, unified set of mechanosensing pathways.

2.
Adv Healthc Mater ; : e2201594, 2022 Nov 18.
Article in English | MEDLINE | ID: mdl-36398536

ABSTRACT

Brain lesions can arise from traumatic brain injury, infection, and craniotomy. Although injectable hydrogels show promise for promoting healing of lesions and health of surrounding tissue, enabling cellular ingrowth and restoring neural tissue continue to be challenging. It is hypothesized that these challenges arise in part from the mismatch of composition, stiffness, and viscoelasticity between the hydrogel and the brain parenchyma, and this hypothesis is tested by developing and evaluating a self-healing hydrogel that not only mimics the composition, but also the stiffness and viscoelasticity of native brain parenchyma. The hydrogel is crosslinked by dynamic boronate ester bonds between phenylboronic acid grafted hyaluronic acid (HA-PBA) and dopamine grafted gelatin (Gel-Dopa). This HA-PBA/Gel-Dopa hydrogel could be injected into a lesion cavity in a shear-thinning manner with rapid hemostasis, high tissue adhesion, and efficient self-healing. In an in vivo mouse model of brain lesions, the multi-functional injectable hydrogel is found to support neural cell infiltration, decrease astrogliosis and glial scars, and close the lesions. The results suggest a role for extracellular matrix-mimicking viscoelasticity in brain lesion healing, and motivate additional experimentation in larger animals as the technology progresses toward potential application in humans.

3.
Nat Commun ; 13(1): 6854, 2022 Nov 11.
Article in English | MEDLINE | ID: mdl-36369425

ABSTRACT

During mesenchymal development, the sources of mechanical forces transduced by cells transition over time from predominantly cell-cell interactions to predominantly cell-extracellular matrix (ECM) interactions. Transduction of the associated mechanical signals is critical for development, but how these signals converge to regulate human mesenchymal stem cells (hMSCs) mechanosensing is not fully understood, in part because time-evolving mechanical signals cannot readily be presented in vitro. Here, we established a DNA-driven cell culture platform that could be programmed to present the RGD peptide from fibronectin, mimicking cell-ECM interactions, and the HAVDI peptide from N-cadherin, mimicking cell-cell interactions, through DNA hybridization and toehold-mediated strand displacement reactions. The platform could be programmed to mimic the evolving cell-ECM and cell-cell interactions during mesenchymal development. We applied this platform to reveal that RGD/integrin ligation promoted cofilin phosphorylation, while HAVDI/N-cadherin ligation inhibited cofilin phosphorylation. Cofilin phosphorylation upregulated perinuclear apical actin fibers, which deformed the nucleus and thereby induced YAP nuclear localization in hMSCs, resulting in subsequent osteogenic differentiation. Our programmable culture platform is broadly applicable to the study of dynamic, integrated mechanobiological signals in development, healing, and tissue engineering.


Subject(s)
Mesenchymal Stem Cells , Osteogenesis , Humans , Integrins/metabolism , Cadherins/metabolism , Phosphorylation , Adhesives/metabolism , Actin Depolymerizing Factors/metabolism , Mesenchymal Stem Cells/metabolism , Cell Differentiation , Extracellular Matrix/metabolism , DNA/metabolism
4.
Small ; : e2204498, 2022 Oct 13.
Article in English | MEDLINE | ID: mdl-36228093

ABSTRACT

Soft-hard tissue interfaces in nature present a diversity of hierarchical transitions in composition and structure to address the challenge of stress concentrations that would otherwise arise at their interface. The translation of these into engineered materials holds promise for improved function of biomedical interfaces. Here, soft-hard tissue interfaces found in the body in health and disease, and the application of the diverse, functionally graded, and hierarchical structures that they present to bioinspired engineering materials are reviewed. A range of such bioinspired engineering materials and associated manufacturing technologies that are on the horizon in interfacial tissue engineering, hydrogel bioadhesion at the interfaces, and healthcare and medical devices are described.

5.
Cell Discov ; 8(1): 84, 2022 Sep 06.
Article in English | MEDLINE | ID: mdl-36068215

ABSTRACT

Cardiac fibrosis is associated with activation of cardiac fibroblasts (CFs), a pathological, phenotypic transition that is widely believed to be irreversible in the late stages of disease development. Sensing of a stiffened mechanical environment through regulation of integrin-based adhesion plaques and activation of the Piezo1 mechanosensitive ion channel is known to factor into this transition. Here, using integrated in vitro and in silico models, we discovered a mutually reinforcing, mechanical positive feedback loop between integrin ß1 and Piezo1 activation that forms a bistable switch. The bistable switch is initiated by perturbations in matrix elastic modulus that amplify to trigger downstream signaling involving Ca2+ and YAP that, recursively, leads fibroblasts to further stiffen their environment. By simultaneously interfering with the newly identified mechanical positive feedback loop and modulating matrix elastic modulus, we reversed markers of phenotypical transition of CF, suggesting new therapeutic targets for fibrotic disease.

6.
Biophys J ; 121(20): 3917-3926, 2022 10 18.
Article in English | MEDLINE | ID: mdl-36045574

ABSTRACT

Acoustic transduction by plants has been proposed as a mechanism to enable just-in-time up-regulation of metabolically expensive defensive compounds. Although the mechanisms by which this "hearing" occurs are unknown, mechanosensation by elongated plant hair cells known as trichomes is suspected. To evaluate this possibility, we developed a theoretical model to evaluate the acoustic radiation force that an elongated cylinder can receive in response to sounds emitted by animals, including insect herbivores, and applied it to the long, cylindrical stem trichomes of the tomato plant Solanum lycopersicum. Based on perturbation theory and validated by finite element simulations, the model quantifies the effects of viscosity and frequency on this acoustic radiation force. Results suggest that acoustic emissions from certain animals, including insect herbivores, may produce acoustic radiation force sufficient to trigger stretch-activated ion channels.


Subject(s)
Animals , Trichomes , Acoustics
7.
Sci Adv ; 8(35): eabn6027, 2022 09 02.
Article in English | MEDLINE | ID: mdl-36044576

ABSTRACT

Chronic kidney diseases are widespread and incurable. The biophysical mechanisms underlying them are unclear, in part because material systems for reconstituting the microenvironment of relevant kidney cells are limited. A critical question is how kidney podocytes (glomerular epithelial cells) regenerate foot processes of the filtration apparatus following injury. Recently identified sarcomere-like structures (SLSs) with periodically spaced myosin IIA and synaptopodin appear in injured podocytes in vivo. We hypothesized that SLSs template synaptopodin in the initial stages of recovery in response to microenvironmental stimuli and tested this hypothesis by developing an ex vivo culture system that allows control of the podocyte microenvironment. Results supported our hypothesis. SLSs in podocytes that migrated from isolated kidney glomeruli presented periodic synaptopodin-positive clusters that nucleated peripheral, foot process-like extensions. SLSs were mechanoresponsive to actomyosin inhibitors and substrate stiffness. Results suggest SLSs as mechanobiological mediators of podocyte recovery and as potential targets for therapeutic intervention.


Subject(s)
Kidney Diseases , Podocytes , Epithelial Cells , Humans , Kidney , Sarcomeres
8.
Nat Mater ; 21(9): 1081-1090, 2022 09.
Article in English | MEDLINE | ID: mdl-35817964

ABSTRACT

How cells sense tissue stiffness to guide cell migration is a fundamental question in development, fibrosis and cancer. Although durotaxis-cell migration towards increasing substrate stiffness-is well established, it remains unknown whether individual cells can migrate towards softer environments. Here, using microfabricated stiffness gradients, we describe the directed migration of U-251MG glioma cells towards less stiff regions. This 'negative durotaxis' does not coincide with changes in canonical mechanosensitive signalling or actomyosin contractility. Instead, as predicted by the motor-clutch-based model, migration occurs towards areas of 'optimal stiffness', where cells can generate maximal traction. In agreement with this model, negative durotaxis is selectively disrupted and even reversed by the partial inhibition of actomyosin contractility. Conversely, positive durotaxis can be switched to negative by lowering the optimal stiffness by the downregulation of talin-a key clutch component. Our results identify the molecular mechanism driving context-dependent positive or negative durotaxis, determined by a cell's contractile and adhesive machinery.


Subject(s)
Actomyosin , Biomechanical Phenomena , Cell Movement
9.
Sci Adv ; 8(23): eabn5460, 2022 Jun 10.
Article in English | MEDLINE | ID: mdl-35675398

ABSTRACT

Transformations in shape are critical to actuation in engineered metamaterials. Existing engineering metamaterials are typically limited to a small number of shape transformations that must be built-in during material synthesis. Here, inspired by the multistability and programmability of kirigami-based self-folding elements, a robust framework is introduced for the construction of sequentially programmable and reprogrammable mechanical metamaterials. The materials can be locked into multiple stable deployed configurations and then, using tunable bistability enabled by temperature-responsive constituent materials, return to their original reference configurations or undergo mode bifurcation. The framework provides a platform to design metamaterials with multiple deployable and reversible configurations in response to external stimuli.

10.
Sci Rep ; 12(1): 5295, 2022 03 28.
Article in English | MEDLINE | ID: mdl-35351922

ABSTRACT

Deep vein thrombosis (DVT) is a potentially deadly medical condition that is costly to treat and impacts thousands of Americans every year. DVT is characterized by the formation of blood clots within the deep venous system of the body. If a DVT dislodges it can lead to venous thromboembolism (VTE) and pulmonary embolism (PE), both of which can lead to significant morbidity or death. Current treatment options for DVT are limited in both effectiveness and safety, in part because the treatment of the DVT cannot be confined to a defined sequestered treatment zone. We therefore developed and tested a novel thrombectomy device that enables the sequesteration of a DVT to a defined treatment zone during fragmentation and evacuation. We observed that, compared to a predicate thrombectomy device, the sequestered approach reduced distal DVT embolization during ex vivo thrombectomy. The sequestered approach also facilitated isovolumetric infusion and suction that enabled clearance of the sequestered treatment zone without significantly impacting vein wall diameter. Results suggest that our novel device using sequestered therapy holds promise for the treatment of high risk large-volume DVTs.


Subject(s)
Pulmonary Embolism , Venous Thromboembolism , Venous Thrombosis , Acute Disease , Humans , Pulmonary Embolism/etiology , Thrombectomy/adverse effects , Venous Thromboembolism/etiology , Venous Thrombosis/etiology , Venous Thrombosis/surgery
11.
Acta Biomater ; 2022 Mar 05.
Article in English | MEDLINE | ID: mdl-35259515

ABSTRACT

The viscoelastic mechanical behavior of collagenous tissues has been studied extensively at the macroscale, yet a thorough quantitative understanding of the time-dependent mechanics of the basic building blocks of tissues, the collagen fibrils, is still missing. In order to address this knowledge gap, stress relaxation and creep tests at various stress (5-35 MPa) and strain (5-20%) levels were performed with individual collagen fibrils (average diameter of fully hydrated fibrils: 253 ± 21 nm) in phosphate buffered saline (PBS). The experimental results showed that the time-dependent mechanical behavior of fully hydrated individual collagen fibrils reconstituted from Type I calf skin collagen, is described by strain-dependent stress relaxation and stress-dependent creep functions in both the heel-toe and the linear regimes of deformation in monotonic stress-strain curves. The adaptive quasilinear viscoelastic (QLV) model, originally developed to capture the nonlinear viscoelastic response of collagenous tissues, provided a very good description of the nonlinear stress relaxation and creep behavior of the collagen fibrils. On the other hand, the nonlinear superposition (NSP) model fitted well the creep but not the stress relaxation data. The time constants and rates extracted from the adaptive QLV and the NSP models, respectively, pointed to a faster rate for stress relaxation than creep. This nonlinear viscoelastic behavior of individual collagen fibrils agrees with prior studies of macroscale collagenous tissues, thus demonstrating consistent time-dependent behavior across length scales and tissue hierarchies. STATEMENT OF SIGNIFICANCE: Pure stress relaxation and creep experiments were conducted for the first time with fully hydrated individual collagen fibrils. It is shown that collagen nanofibrils have a nonlinear time-dependent behavior which agrees with prior studies on macroscale collagenous tissues, thus demonstrating consistent time-dependent behavior across length scales and tissue hierarchies. This new insight into the non-linear viscoelastic behavior of the building blocks of mammalian collagenous tissues may serve as the foundation for improved macroscale tissue models that capture the mechanical behavior across length scales.

12.
Res Sq ; 2022 Feb 21.
Article in English | MEDLINE | ID: mdl-35194598

ABSTRACT

Lateral flow assays (LFAs) are the cornerstone of point-of-care diagnostics. Although rapid and inexpensive, they are 1000-fold less sensitive than laboratory-based tests and cannot be used for definitive negative diagnosis. Here, we overcome this fundamental limitation by employing plasmonically-enhanced nanoscale colorimetric and fluorescent labels. Plasmonic LFAs (p-LFAs) enabled ultrasensitive detection and quantification of low abundance analytes, without compromising the direct visual detection of conventional LFAs. Dynamic ranges and limits of detection were up to 100-fold superior to "gold standard" ELISA (enzyme-linked immunosorbent assay). p-LFAs had sample-to-answer time of 20 min, compared to 4 hours for ELISA, while achieving over 95% analytical sensitivity and 100% analytical specificity for antibodies and antigens of SARS-CoV-2 in human specimens. We also demonstrate that the p-LFAs enable quantitative detection of the target analytes in a standard-free manner. p-LFAs offer potential as a broadly adaptable point-of-care diagnostic platform that outperforms standard laboratory tests in sensitivity, speed, dynamic range, ease of use, and cost.

13.
Adv Healthc Mater ; 11(10): e2102344, 2022 05.
Article in English | MEDLINE | ID: mdl-35026059

ABSTRACT

Current suture-based surgical techniques used to repair torn rotator cuff tendons do not result in mechanically competent tendon-to-bone attachments, leading to high postoperative failure rates. Although adhesives have been proposed to protect against sutures tearing through tendon during healing, no currently available adhesive meets the clinical needs of adhesive strength, biocompatibility, and promotion of healing. Here, a biocompatible, graded, 3,4-dihydroxy phenyl chitosan (BGC) bioadhesive designed to meet these needs is presented. Although 3,4-dihydroxy phenyl chitosan (DP-chitosan) bioadhesives are biocompatible, their adhesion strength is low; soluble oxidants or cross-linking agents can be added for higher bonding strength, but this sacrifices biocompatibility. These challenges are overcome by developing a periodate-modified ion exchange resin-bead filtration system that oxidizes catechol moieties to quinones and filters off the activating agent and resin. The resulting BGC bioadhesive exhibited sixfold higher strength compared to commercially available tissue adhesives, with strength in the range necessary to improve tendon-to-bone repair (≈1MPa, ≈20% of current suture repair strength). The bioadhesive is biocompatible and promoted tenogenesis; cells exposed to the bioadhesive demonstrated enhanced expression of collagen I and the tenogenic marker Scx. Results demonstrated that the bioadhesive has the potential to improve the strength of a tendon-to-bone repair and promote healing.


Subject(s)
Chitosan , Rotator Cuff Injuries , Adhesives , Biomechanical Phenomena , Chitosan/pharmacology , Humans , Rotator Cuff Injuries/metabolism , Rotator Cuff Injuries/surgery , Suture Techniques , Sutures , Tendons/metabolism
14.
ACS Appl Mater Interfaces ; 13(41): 48457-48468, 2021 Oct 20.
Article in English | MEDLINE | ID: mdl-34633172

ABSTRACT

Strong underwater adhesives are attractive materials for biomedical healing and underwater repair, but their success in applications has been limited, owing to challenges with underwater setting and with balancing surface adhesion and cohesion. Here, we applied synthetic biology approaches to overcome these challenges through design and synthesis of a novel hybrid protein consisting of the zipper-forming domains of an amyloid protein, flexible spider silk sequences, and a dihydroxyphenylalanine (DOPA)-containing mussel foot protein (Mfp). This partially structured, hybrid protein can self-assemble into a semi-crystalline hydrogel that exhibits high strength and toughness as well as strong underwater adhesion to a variety of surfaces, including difficult-to-adhere plastics, tendon, and skin. The hydrogel allows selective debonding by oxidation or iron-chelating treatments. Both the material design and the biosynthetic approach explored in this study will inspire future work for a wide range of hybrid protein-based materials with tunable properties and broad applications.


Subject(s)
Adhesives/chemistry , Hydrogels/chemistry , Recombinant Fusion Proteins/chemistry , Adhesiveness , Amyloidogenic Proteins/chemistry , Animals , Bivalvia/chemistry , Fibroins/chemistry , Protein Engineering , Proteins/chemistry , Spiders/chemistry , Viscoelastic Substances/chemistry , Water/chemistry
15.
BMJ Open ; 11(9): e045557, 2021 09 02.
Article in English | MEDLINE | ID: mdl-34475144

ABSTRACT

OBJECTIVE: The COVID-19 pandemic has precipitated widespread shortages of filtering facepiece respirators (FFRs) and the creation and sharing of proposed substitutes (novel designs, repurposed materials) with limited testing against regulatory standards. We aimed to categorically test the efficacy and fit of potential N95 respirator substitutes using protocols that can be replicated in university laboratories. SETTING: Academic medical centre with occupational health-supervised fit testing along with laboratory studies. PARTICIPANTS: Seven adult volunteers who passed quantitative fit testing for small-sized (n=2) and regular-sized (n=5) commercial N95 respirators. METHODS: Five open-source potential N95 respirator substitutes were evaluated and compared with commercial National Institute for Occupational Safety and Health (NIOSH)-approved N95 respirators as controls. Fit testing using the 7-minute standardised Occupational Safety and Health Administration fit test was performed. In addition, protocols that can be performed in university laboratories for materials testing (filtration efficiency, air resistance and fluid resistance) were developed to evaluate alternate filtration materials. RESULTS: Among five open-source, improvised substitutes evaluated in this study, only one (which included a commercial elastomeric mask and commercial HEPA filter) passed a standard quantitative fit test. The four alternative materials evaluated for filtration efficiency (67%-89%) failed to meet the 95% threshold at a face velocity (7.6 cm/s) equivalent to that of a NIOSH particle filtration test for the control N95 FFR. In addition, for all but one material, the small surface area of two 3D-printed substitutes resulted in air resistance that was above the maximum in the NIOSH standard. CONCLUSIONS: Testing protocols such as those described here are essential to evaluate proposed improvised respiratory protection substitutes, and our testing platform could be replicated by teams with similar cross-disciplinary research capacity. Healthcare professionals should be cautious of claims associated with improvised respirators when suggested as FFR substitutes.


Subject(s)
COVID-19 , Occupational Exposure , Respiratory Protective Devices , Adult , Equipment Design , Humans , N95 Respirators , Pandemics/prevention & control , SARS-CoV-2 , United States , Ventilators, Mechanical
16.
J R Soc Interface ; 18(185): 20210421, 2021 12.
Article in English | MEDLINE | ID: mdl-34932930

ABSTRACT

Tendons of the body differ dramatically in their function, mechanics and range of motion, but all connect to bone via an enthesis. Effective force transfer at the enthesis enables joint stability and mobility, with strength and stiffness arising from a fibrous architecture. However, how enthesis toughness arises across tendons with diverse loading orientations remains unclear. To study this, we performed simultaneous imaging of the bone and tendon in entheses that represent the range of tendon-to-bone insertions and extended a mathematical model to account for variations in insertion and bone geometry. We tested the hypothesis that toughness, across a range of tendon entheses, could be explained by differences observed in interactions between fibre architecture and bone architecture. In the model, toughness arose from fibre reorientation, recruitment and rupture, mediated by interactions between fibres at the enthesis and the bony ridge abutting it. When applied to tendons sometimes characterized as either energy-storing or positional, the model predicted that entheses of the former prioritize toughness over strength, while those of the latter prioritize consistent stiffness across loading directions. Results provide insight into techniques for surgical repair of tendon-to-bone attachments, and more broadly into mechanisms for the attachment of highly dissimilar materials.


Subject(s)
Bone and Bones , Tendons , Tendons/diagnostic imaging
17.
Small Methods ; 5(6): e2100276, 2021 06.
Article in English | MEDLINE | ID: mdl-34927916

ABSTRACT

Many cell responses that underlie the development, maturation, and function of tissues are guided by the architecture and mechanical loading of the extracellular matrix (ECM). Because mechanical stimulation must be transmitted through the ECM architecture, the synergy between these two factors is important. However, recapitulating the synergy of these physical microenvironmental cues in vitro remains challenging. To address this, a 3D magnetically actuated collagen hydrogel platform is developed that enables combined control of ECM architecture and mechanical stimulation. With this platform, it is demonstrated how these factors synergistically promote cell alignment of C2C12 myoblasts and enhance myogenesis. This promotion is driven in part by the dynamics of Yes-associated protein and structure of cellular microtubule networks. This facile platform holds great promises for regulating cell behavior and fate, generating a broad range of engineered physiologically representative microtissues in vitro, and quantifying the mechanobiology underlying their functions.


Subject(s)
Extracellular Matrix , Hydrogels , Collagen/chemistry , Hydrogels/pharmacology , Muscle Development , Myoblasts
18.
J Hazard Mater ; 426: 127983, 2022 03 15.
Article in English | MEDLINE | ID: mdl-34923380

ABSTRACT

The trichomes of Arabidopsis thaliana serve as accumulation sites for heavy metals such as Cd2+, and thereby both help plants cope with heavy metal stress and detoxify the soil. These trichomes are also believed to prime plant defenses against insect herbivores in response to mechanical stimulation. Because Cd2+ in such trichomes may be beneficial for plant defenses, we hypothesized that mechanical stimulation would enhance sequestration of Cd2+ in trichomes. We quantified the distribution and concentration of Cd2+ in leaves of A. thaliana, of the glabrous mutant gl1-1 of A. thaliana, and Brassica rapa L. subsp. pekinensis (Lour.) Hanelt (Chinese cabbage) and examined how these changed following mechanical stimulation of the trichomes or leaves. Light brushing or exposure to caterpillars of Spodoptera exigua led trichomes of both A. thaliana and Chinese cabbage to accumulate Cd2+ complexes more rapidly and to a higher concentration than trichomes in unstimulated controls. Comparison to responses in leaves of gl1-1 mutants suggested that this acceleration and enhancement of Cd2+ storage requires signaling through trichomes. In wild type A. thaliana, Cd2+ was found exclusively in trichomes, whereas in gl1-1 mutants, Cd2+ was found mainly in the - mesophyll cells. Results suggest a mechanobiological pathway for improving heavy metal detoxification of soils through the action of hyperaccumulator plant leaves containing non-glandular trichomes.


Subject(s)
Arabidopsis , Brassica , Metals, Heavy , Arabidopsis/genetics , Plant Leaves , Trichomes
19.
Sci Adv ; 7(48): eabi5584, 2021 Nov 26.
Article in English | MEDLINE | ID: mdl-34826240

ABSTRACT

Architectured materials offer tailored mechanical properties but are limited in engineering applications due to challenges in maintaining toughness across their attachments. The enthesis connects tendon and bone, two vastly different architectured materials, and exhibits toughness across a wide range of loadings. Understanding the mechanisms by which this is achieved could inform the development of engineered attachments. Integrating experiments, simulations, and previously unexplored imaging that enabled simultaneous observation of mineralized and unmineralized tissues, we identified putative mechanisms of enthesis toughening in a mouse model and then manipulated these mechanisms via in vivo control of mineralization and architecture. Imaging uncovered a fibrous architecture within the enthesis that controls trade-offs between strength and toughness. In vivo models of pathology revealed architectural adaptations that optimize these trade-offs through cross-scale mechanisms including nanoscale protein denaturation, milliscale load-sharing, and macroscale energy absorption. Results suggest strategies for optimizing architecture for tough bimaterial attachments in medicine and engineering.

20.
J Am Soc Nephrol ; 33(1): 155-173, 2022 01.
Article in English | MEDLINE | ID: mdl-34758982

ABSTRACT

BACKGROUND: Actin stress fibers are abundant in cultured cells, but little is known about them in vivo. In podocytes, much evidence suggests that mechanobiologic mechanisms underlie podocyte shape and adhesion in health and in injury, with structural changes to actin stress fibers potentially responsible for pathologic changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. A technology to image the actin cytoskeleton at high resolution is needed to better understand the role of structures such as actin stress fibers in podocytes. METHODS: We developed the first visualization technique capable of resolving the three-dimensional cytoskeletal network in mouse podocytes in detail, while definitively identifying the proteins that comprise this network. This technique integrates membrane extraction, focused ion-beam scanning electron microscopy, and machine learning image segmentation. RESULTS: Using isolated mouse glomeruli from healthy animals, we observed actin cables and intermediate filaments linking the interdigitated podocyte foot processes to newly described contractile actin structures, located at the periphery of the podocyte cell body. Actin cables within foot processes formed a continuous, mesh-like, electron-dense sheet that incorporated the slit diaphragms. CONCLUSIONS: Our new technique revealed, for the first time, the detailed three-dimensional organization of actin networks in healthy podocytes. In addition to being consistent with the gel compression hypothesis, which posits that foot processes connected by slit diaphragms act together to counterbalance the hydrodynamic forces across the glomerular filtration barrier, our data provide insight into how podocytes respond to mechanical cues from their surrounding environment.


Subject(s)
Actin Cytoskeleton/ultrastructure , Imaging, Three-Dimensional/methods , Machine Learning , Microscopy, Electron , Podocytes/ultrastructure , Animals , Mice , Mice, Inbred C57BL , Models, Animal
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