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Nature Communications Dec 2023The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to...
The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding remain unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of the variants did not affect Vcn activation but reduced Vcn loading and altered exchange dynamics, consistent with the loss of directional catch bonding. Expression of Vcn variants perturbed the coordination of subcellular structures and cell migration, establishing key cellular functions for Vcn directional catch bonding.
Topics: Actins; Vinculin; Actin Cytoskeleton; Cell Movement; Protein Binding
PubMed: 38097542
DOI: 10.1038/s41467-023-43779-x -
Materials Today. Bio Dec 2023Bone resident cells are constantly subjected to a range of distinct mechanical loadings, which generates a complex microenvironment. In particular, hydrostatic pressure...
Bone resident cells are constantly subjected to a range of distinct mechanical loadings, which generates a complex microenvironment. In particular, hydrostatic pressure (HP) has a key impact on modulation of cell function and fate determination. Although HP is a constant mechanical stimulus, its role in regulating the osteogenesis process within a defined 3D microenvironment has not been comprehensively elucidated. Perceiving how environmental factors regulate the differentiation of stem cells is essential for expanding their regenerative potential. Inspired by the mechanical environment of bone, this study attempted to investigate the influence of different ranges of cyclic HP on human adipose-derived mesenchymal stem cells (MSCs) encapsulated within a compartmentalized liquefied microenvironment. Taking advantage of the liquefied environment of microcapsules, MSCs were exposed to cyclic HP of 5 or 50 MPa, 3 times/week at 37 °C. Biological tests using fluorescence staining of F-actin filaments showed a noticeable improvement in cell-cell interactions and cellular network formation of MSCs. These observations were more pronounced in osteogenic (OST) condition, as confirmed by fluorescent staining of vinculin. More interestingly, there was a significant increase in alkaline phosphatase activity of MSCs exposed to 50 MPa magnitude of HP, even in the absence of osteoinductive factors. In addition, a greater staining area of both osteopontin and hydroxyapatite was detected in the 50 MPa/OST group. These findings highlight the benefit of hydrostatic pressure to regulate osteogenesis of MSCs as well as the importance of employing simultaneous biochemical and mechanical stimulation to accelerate the osteogenic potential of MSCs for biomedical purposes.
PubMed: 38058695
DOI: 10.1016/j.mtbio.2023.100861 -
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 -
Regenerative Biomaterials 2023Recombinant humanized collagen (rhCol) was an extracellular matrix (ECM)-inspired biomimetic biomaterial prepared by biosynthesis technology, which was considered...
Recombinant humanized collagen (rhCol) was an extracellular matrix (ECM)-inspired biomimetic biomaterial prepared by biosynthesis technology, which was considered non-allergenic and could possibly activate tissue regeneration. The influence of tag sequence on both structures and performances of rhCol type III (rhCol III) was investigated, and the effect of rhCol III on cell behaviors was evaluated and discussed using Schwann cells (SCs) as model that was critical in the repair process after peripheral nerve injury. The results demonstrated that the introduction of tag sequence would influence both advanced structures and properties of rhCol III, while rhCol III regulated SCs adhesion, spreading, migration and proliferation. Also, both nerve growth factor and brain-derived neurotrophic factor increased when exposed to rhCol III. As the downstream proteins of integrin-mediated cell adhesions, phosphorylation of focal adhesion kinase and expression of vinculin was up-regulated along with the promotion of SCs adhesion and migration. The current findings contributed to a better knowledge of the interactions between rhCol III and SCs, and further offered a theoretical and experimental foundation for the development of rhCol III-based medical devices and clinical management of peripheral nerve injury.
PubMed: 38020236
DOI: 10.1093/rb/rbad089 -
BioRxiv : the Preprint Server For... Oct 2023Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical composition. Their migration has classically been...
Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical composition. 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 protein to migrate efficiently. This migration is mediated through small and dynamic focal adhesions that are composed of the same proteins associated with canonical mesenchymal 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 cell preferentially follows tracks of other T cells, suggesting that these adhesions are modifying 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 adhesions play a key role in T cell motility.
PubMed: 37904911
DOI: 10.1101/2023.10.16.562587 -
Cell Communication and Signaling : CCS Oct 2023Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile...
BACKGROUND
Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile stress could influence cell differentiation through integrin αVβ3.
METHODS
We inhibited the function of integrin αVβ3 of human mesenchymal stem cells by treating with c(RGDyk). Using cytochalasin D and verteporfin to inhibit polymerization of microfilament and function of nuclear Yes-associated protein (YAP), respectively. For each application, mesenchymal stem cells were loaded by cyclic tensile stress of 10% at 0.5 Hz for 2 h daily. Mesenchymal stem cells were harvested on day 7 post-treatment. Western blotting and quantitative RT-PCR were used to detect the expression of alkaline phosphatase (ALP), RUNX2, β-actin, integrin αVβ3, talin-1, vinculin, FAK, and nuclear YAP. Immunofluorescence staining detected vinculin, actin filaments, and YAP nuclear localization.
RESULTS
Cyclic tensile stress could increase the expression of ALP and RUNX2. Inhibition of integrin αVβ3 activation led to rearrangement of actin filaments and downregulated the expression of ALP, RUNX2 and promoted YAP nuclear localization. When microfilament polymerization was inhibited, ALP, RUNX2, and nuclear YAP nuclear localization decreased. Inhibition of YAP nuclear localization could reduce the expression of ALP and RUNX2.
CONCLUSIONS
Cyclic tensile stress promotes early osteogenesis of human mesenchymal stem cells via the integrin αVβ3-actin filaments axis. YAP nuclear localization participates in this process of human mesenchymal stem cells. Video Abstract.
Topics: Humans; Actin Cytoskeleton; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Integrin alphaVbeta3; Mesenchymal Stem Cells; Osteogenesis; Vinculin
PubMed: 37904190
DOI: 10.1186/s12964-022-01027-7 -
The Journal of Cell Biology Jan 2024Vinculin is an actin-binding protein (ABP) that strengthens the connection between the actin cytoskeleton and adhesion complexes. It binds to β-catenin/N-cadherin...
Vinculin is an actin-binding protein (ABP) that strengthens the connection between the actin cytoskeleton and adhesion complexes. It binds to β-catenin/N-cadherin complexes in apical adherens junctions (AJs), which maintain cell-to-cell adhesions, and to talin/integrins in the focal adhesions (FAs) that attach cells to the basal membrane. Here, we demonstrate that β-catenin targets vinculin to the apical AJs and the centrosome in the embryonic neural tube (NT). Suppression of vinculin slows down the basal-to-apical part of interkinetic nuclear migration (BAINM), arrests neural stem cells (NSCs) in the G2 phase of the cell cycle, and ultimately dismantles the apical actin cytoskeleton. In the NSCs, mitosis initiates when an internalized centrosome gathers with the nucleus during BAINM. Notably, our results show that the first centrosome to be internalized is the daughter centrosome, where β-catenin and vinculin accumulate, and that vinculin suppression prevents centrosome internalization. Thus, we propose that vinculin links AJs, the centrosome, and the actin cytoskeleton where actomyosin contraction forces are required.
Topics: Actin Cytoskeleton; Actins; beta Catenin; Cell Adhesion; Cell Cycle; Focal Adhesions; Vinculin; Neural Stem Cells; Centrosome; Adherens Junctions
PubMed: 37889294
DOI: 10.1083/jcb.202106169 -
Journal of Materials Science. Materials... Oct 2023A variety of cell behaviors, such as cell adhesion, motility, and fate, can be controlled by substrate characteristics such as surface topology and chemistry. In...
A variety of cell behaviors, such as cell adhesion, motility, and fate, can be controlled by substrate characteristics such as surface topology and chemistry. In particular, the surface topology of substrates strongly affects cell behaviors, and the topological spacing is a critical factor in inducing cell responses. Various works have demonstrated that cell adhesion was enhanced with decreasing topological spacing although differentiation progressed slowly. However, there are exceptions, and thus, correlations between topological spacing and cell responses are still debated. We show that a nanoporous gold substrate affected cell adhesion while it neither affected osteogenic nor adipogenic differentiation. In addition, the cell adhesion was reduced with decreasing pore size. These do not agree with previous findings. A focal adhesion (FA) is an aggregate of modules comprising specific proteins such as FA kinase, talin, and vinculin. Therefore, it is suggested that because various extracellular signals can be independently branched off from the FA modules, the unusual effects of nanoporous gold substrates are related to the multi-branching of FAs.
Topics: Cell Adhesion; Focal Adhesions; Nanopores; Signal Transduction; Vinculin; Cell Differentiation; Talin; Cell Movement
PubMed: 37884819
DOI: 10.1007/s10856-023-06760-0 -
Cell Reports Nov 2023Focal adhesions (FAs) are dynamic protein assemblies that connect cytoskeletons to the extracellular matrix and are crucial for cell adhesion and migration. KANKs are...
Focal adhesions (FAs) are dynamic protein assemblies that connect cytoskeletons to the extracellular matrix and are crucial for cell adhesion and migration. KANKs are scaffold proteins that encircle FAs and act as key regulators of FA dynamics, but the molecular mechanism underlying their specified localization and functions remains poorly understood. Here, we determine the KANK1 structures in complex with talin and liprin-β, respectively. These structures, combined with our biochemical and cellular analyses, demonstrate how KANK1 scaffolds the FA core and associated proteins to modulate the FA shape in response to mechanical force. Additionally, we find that KANK1 undergoes liquid-liquid phase separation (LLPS), which is important for its localization at the FA edge and cytoskeleton connections to FAs. Our findings not only indicate the molecular basis of KANKs in bridging the core and periphery of FAs but also provide insights into the LLPS-mediated dynamic regulation of FA morphology.
Topics: Focal Adhesions; Protein Binding; Cell Adhesion; Cytoskeleton; Talin
PubMed: 37874676
DOI: 10.1016/j.celrep.2023.113321 -
Fish & Shellfish Immunology Nov 2023Cell migration is an essential process in immunity and wound healing. The in vitro scratch assay was optimized for the SAF-1 cell line, obtained from gilthead seabream...
Cell migration is an essential process in immunity and wound healing. The in vitro scratch assay was optimized for the SAF-1 cell line, obtained from gilthead seabream (Sparus aurata) fin. In addition, selected cells from the cell front were tracked for detailed individual cell movement and morphological analysis. Modulation of migration and cell tracking of the SAF-1 cell line by probiotics was evaluated. Cells were cultured and incubated for 24 h with three species of extremophilic yeasts [Yarrowia lipolytica (D1 and N6) and Debaryomyces hansenii (CBS004)] and the bacterium Shewanella putrefaciens (known as SpPdp11) and then scratch and cell tracking assays were performed. The results indicated that the forward velocity was significantly (p < 0.05) increased in SAF-1 cells incubated with CBS004 or SpPdp11. However, cell velocity, cumulative distance and Euclidean distance were only significantly increased in SAF-1 cells incubated with SpPdp11. Furthermore, to increase our understanding of the genes involved in cell movement, the expression profile of ten structural proteins (α-1β tubulin, vinculin, focal adhesion kinase type, alpha-2 integrin, tetraspanin, integrin-linked kinase 1, tensin 3, tensin 4, paxillin, and light chain 2) was studied by real time-PCR. The expression of these genes was modulated as a function of the probiotic tested and the results indicate that CBS004 and SpPdp11 increase the movement of SAF-1 cells.
Topics: Animals; Sea Bream; Cell Tracking; Tensins; Cell Movement; Probiotics
PubMed: 37858786
DOI: 10.1016/j.fsi.2023.109149