Black dots: High-yield traction force microscopy reveals structural factors contributing to platelet forces

Beussman, Kevin M.; Mollica, Molly Y.; Leonard, Andrea; Miles, Jeffrey D; Hocter, John; Song, Zizhen; Stolla, Moritz; Han, Sangyoon J.; Emery, A. F.; Thomas, Wendy E.; Sniadecki, Nathan J.

doi:10.1016/j.actbio.2021.11.013

Cited by 15 publications

(20 citation statements)

References 42 publications

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“…To test whether changes in marker gene expression were indicative of phenotypic changes in activated fibroblasts, we examined the effect of histone H1.0 depletion on distinct mechanical behaviors in primary cells. We employed a traction force assay, in which cells were seeded onto fluorescently labeled bovine serum albumin (BSA) beads and the deformation of the beads was used to measure the force generated by individual fibroblasts (Beussman et al, 2021). Treatment with TGF- β induced robust traction force generation at the individual cell level and this response was completely abrogated by depletion of histone H1.0 (Figure 2C; Note: In traction force experiments, which measure single cells, all groups were co-transfected with an siRNA conjugated to Cy3 fluorescent tag along with either the scrambled siRNA or the siRNA against histone H1.0, and only cells expressing this tag were selected for measurement).…”

Section: Resultsmentioning

confidence: 99%

“…Primary cardiac fibroblasts transfected with either scrambled or H1.0 siRNA with or without TGF-β stimulation were seeded onto BSA conjugated to a 647-fluorophore micropatterned onto a flexible polydimethylsiloxane (PDMS). Fibroblasts and patterned BSA dots were imaged and deformation of the dots quantified and converted into forces as described (Beussman et al, 2021).…”

Section: Traction Force Assaymentioning

confidence: 99%

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Histone H1.0 Couples Cellular Mechanical Behaviors to Chromatin Structure

Chapski

Gehred

et al. 2022

Preprint

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Tuning of genome structure and function is accomplished by chromatin binding proteins, which determine the transcriptome and phenotype of the cell. We sought to investigate how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors. We demonstrate that the linker histone H1.0, which compacts nucleosomes into higher order chromatin fibers, controls genome organization and cellular stress response. Histone H1.0 has privileged expression in fibroblasts across tissue types in mice and humans, and modulation of its expression is necessary and sufficient to mount a myofibroblast phenotype in these cells. Depletion of histone H1.0 prevents transforming growth factor beta (TGF-beta)-induced fibroblast contraction, proliferation and migration in a histone H1 isoform-specific manner via inhibition of a transcriptome comprised of extracellular matrix, cytoskeletal and contractile genes. Histone H1.0 is associated with local regulation of gene expression via mechanisms involving chromatin fiber compaction and reprogramming of histone acetylation, rendering the cell stiffer in response to cytokine stimulation. Knockdown of histone H1.0 prevented locus-specific histone H3 lysine 27 acetylation by TGF-beta and decreased levels of both HDAC1 and the chromatin reader BRD4, thereby preventing transcription of a fibrotic gene program. Transient depletion of histone H1.0 in vivo decompacts chromatin and prevents fibrosis in cardiac muscle, thereby linking chromatin structure with fibroblast phenotype in response to extracellular stress. Our work identifies an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling cellular force generation, nuclear organization and gene transcription.

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Section: Resultsmentioning

confidence: 99%

Section: Traction Force Assaymentioning

confidence: 99%

Histone H1.0 Couples Cellular Mechanical Behaviors to Chromatin Structure

Chapski

Gehred

et al. 2022

Preprint

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“…Measuring the contractility of single platelets is informative because it can reveal heterogeneity among the platelet population; however, it is experimentally demanding. Techniques yielding absolute single cell force values range from AFM [65], over hydrogels [49,55,66], micropatterned hydrogels, [50,56], negatively micropatterned elastomers [42 ▪ ,67 ▪ ,68], to micropost arrays [43 ▪ ,44,57 ▪▪ ], as summarized in Fig. 3 and described elsewhere [69].…”

Section: Platelet Contractility As a Hemostatic Parameter?mentioning

confidence: 99%

Platelet mechanosensing as key to understanding platelet function

Schoen,

Kenny,

Patil

2023

Current Opinion in Hematology

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Purpose of review This review highlights how the perception of platelet function is evolving based on recent insights into platelet mechanobiology. Recent findings The mechanosensitive ion channel Piezo1 mediates activation of free-flowing platelets under conditions of flow acceleration through mechanisms independent of adhesion receptors and classical activation pathways. Interference with the initiation of platelet migration or with the phenotypic switch of migrating platelets to a procoagulant state aggravates inflammatory bleeding. Mechanosensing of biochemical and biophysical microenvironmental cues during thrombus formation feed into platelet contractile force generation. Measurements of single platelet contraction and bulk clot retraction show promise to identify individuals at risk for hemorrhage. Summary New findings unravel novel mechanotransduction pathways and effector functions in platelets, establishing mechanobiology as a pivotal component of platelet function. These insights highlight limitations of existing treatments and offer new potential therapeutic approaches and diagnostic avenues based on mechanobiological principles. Further extensive research is required to distinguish between core hemostatic and pathological mechanisms influenced by platelet mechanosensing.

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“…Typically, regularly arranged fluorescent dots or pillars formed the foundation for reference-free TFMs. [26][27][28][29] These reference-free TFMs provided an approachable technique for sensing cellular contractility during cell growth. However, the tedious preparation process and the request of precious instruments including focused laser beam or electron-beam lithography limited their broad applications.…”

Section: Introductionmentioning

confidence: 99%