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Nordic Journal of Psychiatry Jul 2023Increased intestinal and blood-brain barriers (BBB) permeability has been suggested to have a role in autism spectrum disorder (ASD). Claudin-5, claudin-11, occludin,...
AIM
Increased intestinal and blood-brain barriers (BBB) permeability has been suggested to have a role in autism spectrum disorder (ASD). Claudin-5, claudin-11, occludin, β-catenin, vinculin, and paxillin are crucial components of these barriers. This study assessed concentrations of these molecules in preschool children with ASD.
METHODS
A total of 80 children with ASD and 40 controls aged 18-60 months were enrolled in this study. Serum levels of biochemical variables were determined using commercial enzyme-linked immunosorbent assay kits.
RESULTS
Serum claudin-11, occludin, and β-catenin levels were significantly higher in the ASD group than in the control group. However, no significant difference for serum claudin-5, vinculin, and paxillin levels was detected between the groups.
CONCLUSION
These findings suggest that claudin-11, occludin, and β-catenin may be involved in the pathogenesis of ASD. These proteins may affect the brain by causing dysregulation in intestinal or blood-brain barrier permeability or with other unknown mechanisms.
Topics: Child, Preschool; Humans; Infant; Autism Spectrum Disorder; beta Catenin; Biomarkers; Claudin-5; Claudins; Occludin; Paxillin; Vinculin; Blood-Brain Barrier; Permeability; Intestines
PubMed: 36662163
DOI: 10.1080/08039488.2023.2168055 -
PeerJ 2024Calcium (Ca) homeostasis is essential in conducting various cellular processes including nerve transmission, muscular movement, and immune response. Changes in...
Calcium (Ca) homeostasis is essential in conducting various cellular processes including nerve transmission, muscular movement, and immune response. Changes in Ca concentration in the cytoplasm are significant in bringing about various immune responses such as pathogen clearance and apoptosis. Various key players are involved in calcium homeostasis such as calcium binders, pumps, and channels. Sequence-based evolutionary information has recently been exploited to predict the biophysical behaviors of proteins, giving critical clues about their functionality. Ion channels are reportedly the first channels developed during evolution. Calcium homeostasis modulator protein 6 (CALHM6) is one such channel. Comprised of a single domain called Ca_hom_mod, CALHM6 is a stable protein interacting with various other proteins in calcium regulation. No previous attempt has been made to trace the exact evolutionary events in the domain of CALHM6, leaving plenty of room for exploring its evolution across a wide range of organisms. The current study aims to answer the questions by employing a computational-based strategy that used profile Hidden Markov Models (HMMs) to scan for the CALHM6 domain, integrated the data with a time-calibrated phylogenetic tree using BEAST and Mesquite, and visualized through iTOL. Around 4,000 domains were identified, and 14,000 domain gain, loss, and duplication events were observed at the end which also included various protein domains other than CALHM6. The data were analyzed concerning CALHM6 evolution as well as the domain gain, loss, and duplication of its interacting partners: Calpain, Vinculin, protein S100-A7, Thioredoxin, Peroxiredoxin, and Calmodulin-like protein 5. Duplication events of CALHM6 near higher eukaryotes showed its increasing complexity in structure and function. This phylogenetic approach applied to trace the evolution of CALHM6 was an effective approach to get a better understanding of the protein CALHM6.
Topics: Phylogeny; Protein Domains; Bone Density Conservation Agents; Calcium, Dietary; Homeostasis; Hormone Antagonists
PubMed: 38188152
DOI: 10.7717/peerj.16063 -
Biophysical Journal Dec 2023Transmission of cell-generated (i.e., endogenous) tension at cell-cell contacts is crucial for tissue shape changes during morphogenesis and adult tissue repair in...
Transmission of cell-generated (i.e., endogenous) tension at cell-cell contacts is crucial for tissue shape changes during morphogenesis and adult tissue repair in tissues such as epithelia. E-cadherin-based adhesions at cell-cell contacts are the primary means by which endogenous tension is transmitted between cells. The E-cadherin-β-catenin-α-catenin complex mechanically couples to the actin cytoskeleton (and thereby the cell's contractile machinery) both directly and indirectly. However, the key adhesion constituents required for substantial endogenous force transmission at these adhesions in cell-cell contacts are unclear. Due to the role of α-catenin as a mechanotransducer that recruits vinculin at cell-cell contacts, we expected α-catenin to be essential for sustaining normal levels of force transmission. Instead, using the traction force imbalance method to determine the inter-cellular force at a single cell-cell contact between cell pairs, we found that it is vinculin that is essential for sustaining normal levels of endogenous force transmission, with absence of vinculin decreasing the inter-cellular tension by over 50%. Our results constrain the potential mechanical pathways of force transmission at cell-cell contacts and suggest that vinculin can transmit forces at E-cadherin adhesions independent of α-catenin, possibly through β-catenin. Furthermore, we tested the ability of lateral cell-cell contacts to withstand external stretch and found that both vinculin and α-catenin are essential to maintain cell-cell contact stability under external forces.
Topics: alpha Catenin; Vinculin; beta Catenin; Cadherins; Cell Adhesion; Actins
PubMed: 38350000
DOI: 10.1016/j.bpj.2023.10.029 -
Matrix Biology : Journal of the... Aug 2023Basement membranes (BMs) are critical but frequently ignored components of the vascular system. Using high-resolution confocal imaging of whole-mount-stained mesenteric...
Basement membranes (BMs) are critical but frequently ignored components of the vascular system. Using high-resolution confocal imaging of whole-mount-stained mesenteric arteries, we identify integrins, vinculin, focal adhesion kinase (FAK) and several BM proteins including laminins as novel components of myoendothelial junctions (MEJs), anatomical microdomains that are emerging as regulators of cross-talk between endothelium and smooth muscle cells (SMCs). Electron microscopy revealed multiple layers of the endothelial BM that surround endothelial projections into the smooth muscle layer as structural characteristics of MEJs. The shear-responsive calcium channel TRPV4 is broadly distributed in endothelial cells and occurs in a proportion of MEJs where it localizes to the tips of the endothelial projections that are in contact with the underlying SMCs. In mice lacking the major endothelial laminin isoform, laminin 411 (Lama4), which we have previously shown over-dilate in response to shear and exhibit a compensatory laminin 511 upregulation, localization of TRPV4 at the endothelial-SMC interface in MEJs was increased. Endothelial laminins do not affect TRPV4 expression, rather in vitro electrophysiology studies using human umbilical cord arterial endothelial cells revealed enhanced TRPV4 signalling upon culturing on an RGD-motif containing domain of laminin 511. Hence, integrin-mediated interactions with laminin 511 in MEJ structures unique to resistance arteries modulate TRPV4 localization at the endothelial-smooth muscle interface in MEJs and signalling over this shear-response molecule.
Topics: Mice; Humans; Animals; Laminin; Endothelial Cells; TRPV Cation Channels; Basement Membrane; Endothelium, Vascular; Communication
PubMed: 37311512
DOI: 10.1016/j.matbio.2023.06.001 -
ACS Biomaterials Science & Engineering Apr 2024Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such...
Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or -(2-hydroxyethyl)piperazine--ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.
Topics: Collagen; Extracellular Matrix; Macrophages; Transcription Factors; Hydrogels; Cytokines
PubMed: 38467019
DOI: 10.1021/acsbiomaterials.3c01892 -
Life Science Alliance Aug 2024Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in , encoding the first step in the synthesis...
Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in , encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in mice there is a failure of the α7β1 integrin complex that is specific to affected muscle. We observed that in hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of .
Topics: Animals; Mice; Vinculin; Mice, Knockout; Muscular Dystrophies; Integrins; Choline Kinase; Sarcolemma; Humans; Focal Adhesions; Cell Membrane; Actinin; Muscle, Skeletal; Phosphatidylinositol 4,5-Diphosphate; Actins; Disease Models, Animal
PubMed: 38749543
DOI: 10.26508/lsa.202301956 -
Proceedings of the National Academy of... Dec 2023The ability of cells to move in a mechanically coupled, coordinated manner, referred to as collective cell migration, is central to many developmental, physiological,...
The ability of cells to move in a mechanically coupled, coordinated manner, referred to as collective cell migration, is central to many developmental, physiological, and pathophysiological processes. Limited understanding of how mechanical forces and biochemical regulation interact to affect coupling has been a major obstacle to unravelling the underlying mechanisms. Focusing on the linker protein vinculin, we use a suite of Förster resonance energy transfer-based biosensors to probe its mechanical functions and biochemical regulation, revealing a switch that toggles vinculin between loadable and unloadable states. Perturbation of the switch causes covarying changes in cell speed and coordination, suggesting alteration of the friction within the system. Molecular scale modelling reveals that increasing levels of loadable vinculin increases friction, due to engagement of self-stabilizing catch bonds. Together, this work reveals a regulatory switch for controlling cell coupling and describes a paradigm for relating biochemical regulation, altered mechanical properties, and changes in cell behaviors.
Topics: Vinculin; Cell Movement; Mechanical Phenomena; Fluorescence Resonance Energy Transfer; Cell Adhesion
PubMed: 38055737
DOI: 10.1073/pnas.2316456120 -
Biomacromolecules Dec 2023In-depth understanding of the mechanisms underlying the adhesion of myocardial cells holds significant importance for the development of effective therapeutic...
In-depth understanding of the mechanisms underlying the adhesion of myocardial cells holds significant importance for the development of effective therapeutic biomaterials aimed at repairing damaged or pathological myocardial tissues. Herein, we present evidence that myocardial cells (H9C2) exhibit integrin-based mechanosensing during the initial stage of adhesion (within the first 2 h), enabling them to recognize and respond to variations in substrate stiffnesses. Moreover, the bioinformatics analysis of RNA transcriptome sequencing (RNA-seq) reveals that the gene expressions associated with initial stage focal adhesion (Ctgf, Cyr61, Amotl2, Prickle1, Serpine1, Akap12, Hbegf, and Nedd9) are up-regulated on substrates with elevated Young's modulus. The fluorescent immunostaining results also suggest that increased substrate stiffness enhances the expression of Y397-phosphorylated focal adhesion kinase (FAK Y397), talin, and vinculin and the assembly of F-actin in H9C2 cells, thereby facilitating the adhesion of myocardial cells on the substrate. Next, we utilize fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS) to quantitatively evaluate the impact of substrate stiffness on the cell adhesion force and adhesion work, thus providing novel insights into the biomechanical regulation of initial cell adhesion. Our findings demonstrate that the maximum adhesion forces of myocardial cells exhibit a rise from 23.6 to 248.0 nN when exposed to substrates with different moduli. It is worth noting that once the αvβ3 integrins are blocked, the disparities in the adhesion forces of myocardial cells on these substrates become negligible. These results exhibit remarkable sensitivity of myocardial cells to mechanical cues of the substrate, highlighting the role of αvβ3 integrin as a biomechanical sensor for the regulation of cell adhesion. Overall, this work offers a prospective approach for the regulation of cell adhesion via integrin mechanosensing with potential practical applications in the areas of tissue engineering and regenerative medicine.
Topics: Myocytes, Cardiac; Cues; Cell Adhesion; Integrins; Focal Adhesions
PubMed: 37956199
DOI: 10.1021/acs.biomac.3c00871 -
Stem Cell Research & Therapy Feb 2024Pericytes (PCs) are multipotent contractile cells that wrap around the endothelial cells (ECs) to maintain the blood vessel's functionality and integrity. The...
BACKGROUND
Pericytes (PCs) are multipotent contractile cells that wrap around the endothelial cells (ECs) to maintain the blood vessel's functionality and integrity. The hyperglycemia associated with Type 2 diabetes mellitus (T2DM) was shown to impair the function of PCs and increase the risk of diabetes complications. In this study, we aimed to investigate the deleterious effect of the diabetic microenvironment on the regenerative capacities of human PCs.
METHODS
PCs isolated from human adipose tissue were cultured in the presence or absence of serum collected from diabetic patients. The functionality of PCs was analyzed after 6, 14, and 30 days.
RESULTS
Microscopic examination of PCs cultured in DS (DS-PCs) showed increased aggregate formation and altered surface topography with hyperbolic invaginations. Compared to PCs cultured in normal serum (NS-PCs), DS-PCs showed more fragmented mitochondria and thicker nuclear membrane. DS caused impaired angiogenic differentiation of PCs as confirmed by tube formation, decreased VEGF-A and IGF-1 gene expression, upregulated TSP1, PF4, actin-related protein 2/3 complex, and downregulated COL21A1 protein expression. These cells suffered more pronounced apoptosis and showed higher expression of Clic4, apoptosis facilitator BCl-2-like protein, serine/threonine protein phosphatase, and caspase-7 proteins. DS-PCs showed dysregulated DNA repair genes CDKN1A, SIRT1, XRCC5 TERF2, and upregulation of the pro-inflammatory genes ICAM1, IL-6, and TNF-α. Further, DS-treated cells also showed disruption in the expression of the focal adhesion and binding proteins TSP1, TGF-β, fibronectin, and PCDH7. Interestingly, DS-PCs showed resistance mechanisms upon exposure to diabetic microenvironment by maintaining the intracellular reactive oxygen species (ROS) level and upregulation of extracellular matrix (ECM) organizing proteins as vinculin, IQGAP1, and tubulin beta chain.
CONCLUSION
These data showed that the diabetic microenvironment exert a deleterious effect on the regenerative capacities of human adipose tissue-derived PCs, and may thus have possible implications on the vascular complications of T2DM. Nevertheless, PCs have shown remarkable protective mechanisms when initially exposed to DS and thus they could provide a promising cellular therapy for T2DM.
Topics: Humans; Diabetes Mellitus, Type 2; Pericytes; Endothelial Cells; Adipose Tissue; Apoptosis; Cells, Cultured
PubMed: 38331889
DOI: 10.1186/s13287-024-03643-1 -
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