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BioRxiv : the Preprint Server For... Jun 2024Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes...
Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.
PubMed: 38895381
DOI: 10.1101/2024.06.06.597759 -
The Journal of Cell Biology Oct 2024Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical compositions. Their migration has classically been...
Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical compositions. Their migration has classically been defined as amoeboid under the assumption that it is integrin independent. Here, we show that activated primary Th1 T cells require both confinement and extracellular matrix proteins to migrate efficiently. This migration is mediated through small and dynamic focal adhesions that are composed of the same proteins associated with canonical mesenchymal cell focal adhesions, such as integrins, talin, and vinculin. These focal adhesions, furthermore, localize to sites of contractile traction stresses, enabling T cells to pull themselves through confined spaces. Finally, we show that Th1 T cells preferentially follow tracks of other T cells, suggesting that these adhesions modify the extracellular matrix to provide additional environmental guidance cues. These results demonstrate not only that the boundaries between amoeboid and mesenchymal migration modes are ambiguous, but that integrin-mediated focal adhesions play a key role in T cell motility.
Topics: Focal Adhesions; Animals; Cell Movement; Integrins; Talin; Mice; Extracellular Matrix; Vinculin; Mice, Inbred C57BL; T-Lymphocytes; Lymphocyte Activation; Cell Adhesion
PubMed: 38889096
DOI: 10.1083/jcb.202310067 -
Allergy Jun 2024The updated World Health Organization (WHO) air quality guideline recommends an annual mean concentration of fine particulate matter (PM2.5) not exceeding 5 or...
Comprehensive analysis of resilience of human airway epithelial barrier against short-term PM2.5 inorganic dust exposure using in vitro microfluidic chip and ex vivo human airway models.
BACKGROUND AND OBJECTIVE
The updated World Health Organization (WHO) air quality guideline recommends an annual mean concentration of fine particulate matter (PM2.5) not exceeding 5 or 15 μg/m in the short-term (24 h) for no more than 3-4 days annually. However, more than 90% of the global population is currently exposed to daily concentrations surpassing these limits, especially during extreme weather conditions and due to transboundary dust transport influenced by climate change. Herein, the effect of respirable
METHODS
Silica particles at an average size of 1 μm, referred to as
RESULTS
In the AEB-on-a-chip platform, short-term exposure to 800 μg/mL PM2.5 disrupted AEB integrity via increasing barrier permeability, decreasing cell adhesion-barrier markers such as ZO-1, Vinculin, ACE2, and CD31, impaired cell viability and increased the expression levels of proinflammatory markers; IFNs, IL-6, IL-1s, TNF-α, CD68, CD80, and Inos, mostly under dynamic conditions. Besides, decreased tissue viability, impaired tissue integrity via decreasing of Vinculin, ACE2, β-catenin, and E-cadherin, and also proinflammatory response with elevated CD68, IL-1α, IL-6, IFN-Ɣ, Inos, and CD80 markers, were observed after PM2.5 exposure in ex vivo tissue.
CONCLUSION
The duration and concentration of PM2.5 that can be exposed during extreme weather conditions and natural events aligns with our exposure model (0-800 μg/mL 72 h). At this level of exposure, the resilience of the epithelial barrier is demonstrated by both AEB-on-a-chip platform emulating dynamic forces in the body and ex vivo bronchial biopsy slices. Lung-on-a-chip models will serve as reliable exposure models in this context.
PubMed: 38868934
DOI: 10.1111/all.16179 -
Nature Communications Jun 2024Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well...
Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.
Topics: Talin; Focal Adhesions; Cell Membrane; Vinculin; Humans; Animals; Phosphatidylinositol 4,5-Diphosphate; Integrins; Cytoplasm; Protein Binding; Phase Separation
PubMed: 38862544
DOI: 10.1038/s41467-024-49222-z -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... May 2024To evaluate the therapeutic effect of normal mouse serum on radiation pneumonitis in mice and explore the possible mechanism.
OBJECTIVE
To evaluate the therapeutic effect of normal mouse serum on radiation pneumonitis in mice and explore the possible mechanism.
METHODS
Mouse models of radiation pneumonitis induced by thoracic radiation exposure were given intravenous injections of 100 μL normal mouse serum or normal saline immediately after the exposure followed by injections once every other day for a total of 8 injections. On the 15th day after irradiation, histopathological changes of the lungs of the mice were examined using HE staining, the levels of TNF-α, TGF-β, IL-1α and IL-6 in the lung tissue and serum were detected using ELISA, and the percentages of lymphocytes in the lung tissue were analyzed with flow cytometry. High-throughput sequencing of exosome miRNA was carried out to explore the changes in the signaling pathways. The mRNA expression levels of the immune-related genes were detected by qRT-PCR, and the protein expressions of talin-1, tensin2, FAK, vinculin, α-actinin and paxillin in the focal adhesion signaling pathway were detected with Western blotting.
RESULTS
In the mouse models of radiation pneumonitis, injections of normal mouse serum significantly decreased the lung organ coefficient, lowered the levels of TNF-α, TGF-β, IL-1α and IL-6 in the serum and lung tissues, and ameliorated infiltration of CD45, CD4 and T lymphocytes in the lung tissue (all <0.05). The expression levels of and genes at both the mRNA and protein levels and the protein expressions of talin-1, tensin2, FAK, vinculin, α‑actinin and paxillin were all significantly down-regulated in the mouse models after normal mouse serum treatment.
CONCLUSION
Normal mouse serum ameliorates radiation pneumonitis in mice by inhibiting the expressions of key proteins in the Focal adhesion signaling pathway.
Topics: Animals; Mice; Signal Transduction; Radiation Pneumonitis; Focal Adhesions; Lung; Interleukin-6; Disease Models, Animal; Tumor Necrosis Factor-alpha; Transforming Growth Factor beta; MicroRNAs; Interleukin-1alpha
PubMed: 38862437
DOI: 10.12122/j.issn.1673-4254.2024.05.01 -
Se Pu = Chinese Journal of... Jun 2024Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The...
Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The disruption of mitochondrial bioenergetic and metabolic functions is closely related to many disorders. The specific isolation and purification of intact, high-purity, and functional mitochondria are central to the understanding of their mechanism of action but remain challenging tasks. In this study, a mitochondrial penetrating peptide (MPP) with the sequence FrFKFrFK(Ac) was used as a mitochondrial recognition motif to construct a peptide-guided affinity separation material. The multiple aromatic phenylalanine (F) residues in this amphiphilic peptide can confer lipophilicity to the mitochondrial membrane, whereas the basic residues (D-arginine and lysine) render the MPP surface positively charged, thereby promoting the binding of negatively charged mitochondria. After the derivatization of the N terminal of MPP with an oligoglycine spacer, the peptide ligands were conjugated to matrix beads (MB) with surface aldehyde functional groups. Peptide functionalization was performed via a condensation reaction between the amino group in the peptide ligand and the aldehyde group on the beads. The generated Schiff bases were reduced, affording stable covalent bonds. The dense and stable functionalization of the beads with the mitochondria-targeting peptides was demonstrated using high performance liquid chromatography (HPLC), zeta potential assay, and scanning electron microscopy (SEM). The immobilization efficiency of the peptide ligands was 1.47 μmol/g, and the surface potential of MB@MPP was 11 mV. MB@MPP was used for the direct isolation of mitochondria after cell homogenization. As observed by SEM, mitochondria with a cross-sectional diameter of 500 nm were efficiently captured on the MB@MPP surface. Because the mitochondrial membrane potential is an important marker of mitochondrial function and the driving force behind the staining of mitochondria with Mito Tracker dyes, the specific binding and separation of fluorescent mitochondria from the cell samples revealed that the proposed MB@MPP-based isolation approach can keep mitochondria intact and retain their functions. Western blot assays were employed to characterize the protein markers of the mitochondria (citrate synthase (CS) and voltage-dependent anion channel protein (VDAC)) and cytoplasmic protein (vinculin), and examine the integrity and purity of the captured mitochondria. The results showed that the lysates released from MB@MPP had high CS and VDAC contents. By contrast, vinculin, which is highly abundant in whole-cell lysates, was barely detected in the lysates from MB@MPP. These results suggest that MB@MPP isolates mitochondria with high affinity, specificity, and antifouling ability by using the targeting peptide as the capture handle. A comparison with a commercial mitochondrial isolation kit demonstrated that MB@MPP can separate mitochondria with higher CS and VDAC abundance and purity. Given the superior separation performance of MB@MPP, the molecular profiles of the isolated mitochondria under stress were subjected to further analysis of their molecular profiles under stress. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to detect tryptophan (Trp) and riboflavin in the mitochondria. Quantification was performed in multiple-reaction monitoring (MRM) mode. Owing to the high purity of the mitochondria, the Trp and riboflavin contents were determined to be 265 and 0.67 nmol/mg, respectively. The metabolic response of mitochondria to external stimuli was further examined using acadesine, an adenosine 5'-monophosphate (AMP)-activated protein kinase activator with a wide range of metabolic effects, to treat cells. After cell homogenization, MB@MPP was used to separate the mitochondria from the cell samples with and without acadesine treatment, followed by LC-MS/MS analysis. The quantification results demonstrated that acadesine induced a 14% upregulation of Trp content in the mitochondria. By contrast, the riboflavin content decreased to 0.48 nmol/mg, which is 72% of that in untreated mitochondria. The changes in Trp and riboflavin contents could influence their metabolic pathways and, thus, the levels of their metabolites, such as nicotinamide adenine dinucleotide, flavin mononucleotide, and flavin adenine dinucleotide, which are essential coenzymes in mitochondria. Peptide-functionalized affinity microbeads with high affinity and specificity for mitochondria are promising for the efficient isolation of high-quality mitochondria, and offer a useful tool for understanding the complicated functions and dynamics of this unique organelle.
Topics: Mitochondria; Peptides; Animals; Chromatography, Affinity
PubMed: 38845516
DOI: 10.3724/SP.J.1123.2024.01013 -
Cell Jun 2024Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and...
Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.
Topics: Mechanotransduction, Cellular; Humans; Single Molecule Imaging; Animals; DNA; Focal Adhesions; Integrins; Microscopy, Atomic Force; Mice; Cell Adhesion; Biomechanical Phenomena
PubMed: 38838668
DOI: 10.1016/j.cell.2024.05.008 -
Life Science Alliance Aug 2024Vinculin is a cytoskeletal linker strengthening cell adhesion. The IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with...
Vinculin is a cytoskeletal linker strengthening cell adhesion. The IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain-mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains' residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a and D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell-matrix adhesion.
Topics: Vinculin; Humans; Focal Adhesions; Cell Adhesion; Bacterial Proteins; Mutation; Host-Pathogen Interactions; HeLa Cells; Protein Binding; Shigella; Antigens, Bacterial; Dysentery, Bacillary
PubMed: 38834194
DOI: 10.26508/lsa.202302418 -
American Journal of Obstetrics and... May 2024Black women experience a disproportionate impact of uterine fibroids compared to White women, including earlier diagnosis, higher frequency, and more severe symptoms....
BACKGROUND
Black women experience a disproportionate impact of uterine fibroids compared to White women, including earlier diagnosis, higher frequency, and more severe symptoms. The etiology underlying this racial disparity remains elusive.
OBJECTIVE
The aim of this study was to evaluate the molecular differences in normal myometrium (fibroid-free uteri) and at-risk myometrium (fibroid-containing uteri) tissues in Black and White women.
STUDY DESIGN
We conducted whole-genome RNA-seq on normal and at-risk myometrium tissues obtained from both self-identified Black and White women (not Hispanic or Latino) to determine global gene expression profiles and to conduct enriched pathway analyses (n=3 per group). We initially assessed the differences within the same type of tissue (normal or at-risk myometrium) between races. Subsequently, we analyzed the transcriptome of normal myometrium compared to at-risk myometrium in each race and determined the differences between them. We validated our findings through real-time PCR (sample size range=5-12), western blot (sample size range=5-6), and immunohistochemistry techniques (sample size range=9-16).
RESULTS
The transcriptomic analysis revealed distinct profiles between Black and White women in normal and at-risk myometrium tissues. Interestingly, genes and pathways related to extracellular matrix and mechanosensing were more enriched in normal myometrium from Black than White women. Transcription factor enrichment analysis detected greater activity of the serum response transcription factor positional motif in normal myometrium from Black compared to White women. Furthermore, we observed increased expression levels of myocardin-related transcription factor-serum response factor and the serum response factor in the same comparison. In addition, we noted increased expression of both mRNA and protein levels of vinculin, a target gene of the serum response factor, in normal myometrium tissues from Black women as compared to White women. Importantly, the transcriptomic profile of normal to at-risk myometrium conversion differs between Black and White women. Specifically, we observed that extracellular matrix-related pathways are involved in the transition from normal to at-risk myometrium and that these processes are exacerbated in Black women. We found increased levels of Tenascin C, type I collagen alpha 1 chain, fibronectin and, phospho-p38 MAPK (Thr180/Tyr182, active) protein levels in at-risk over normal myometrium tissues from Black women, whereas such differences were not observed in samples from White women.
CONCLUSIONS
These findings indicate that the racial disparities in uterine fibroids may be attributed to heightened production of extracellular matrix in the myometrium in Black women, even before the tumors appear. Future research is needed to understand early life determinants of the observed racial differences.
PubMed: 38825029
DOI: 10.1016/j.ajog.2024.05.048 -
European Journal of Cell Biology May 2024Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and...
Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and transducer by recruiting vinculin and transducing signals that influence cell behaviors. α-catenin/vinculin complex-mediated mechanotransduction regulates multiple pathways, such as Hippo pathway. However, their associations with the α-catenin-based tension sensors at cell junctions are still not fully addressed. Here, we uncovered the TRIP6/LATS1 complex co-localizes with α-catenin/vinculin at both bicellular junctions (BCJs) and tricellular junctions (TCJs). The localization of TRIP6/LATS1 complex to both TCJs and BCJs requires ROCK1 and α-catenin. Treatment by cytochalasin B, Y-27632 and blebbistatin all impaired the BCJ and TCJ junctional localization of TRIP6/LATS1, indicating that the junctional localization of TRIP6/LATS1 is mechanosensitive. The α-catenin/vinculin/TRIP6/LATS1 complex strongly localized to TCJs and exhibited a discontinuous button-like pattern on BCJs. Additionally, we developed and validated an α-catenin/vinculin BiFC-based mechanosensor that co-localizes with TRIP6/LATS1 at BCJs and TCJs. The mechanosensor exhibited a discontinuous distribution and motile signals at BCJs. Overall, our study revealed that TRIP6 and LATS1 are novel compositions of the tension sensor, together with the core complex of α-catenin/vinculin, at both the BCJs and TCJs.
PubMed: 38805800
DOI: 10.1016/j.ejcb.2024.151426