Optical tweezers combined with FRET tension sensor reveal force-dependent vinculin dynamics

bioRxiv (Cold Spring Harbor Laboratory) · 2025-11-10

Abstract Methods to visualize and quantify the molecular responses of cells to local forces exerted at adhesions are crucial to elucidate how physical forces control cellular behavior. Of the many proteins involved in focal adhesions, vinculin plays a key role in mediating force-sensitive processes. Here, we combined optical tweezers and Förster resonance energy transfer (FRET) microscopy to measure the intensity and FRET efficiency of the vinculin tension sensor, VinTS, in response to a force. Fibroblasts expressing VinTS formed adhesions on fibronectin-coated, 3□m-diameter, polystyrene beads. As the beads were displaced by the cell, we applied an optical trap to counteract this movement and increase the traction force required by the cell to maintain the bead’s displacement. The optical trap stiffness varied from zero (no laser) up to 0.26 pN/nm. In this range, the median bead displacement after 5 min was ∼200nm in all trapping conditions inducing counteracting forces in the 10-100pN range. To maintain this displacement, vinculin recruitment increased (up to 35% in relative intensity at high stiffness) while tension increased but more moderately (1-2% decrease in absolute FRET efficiency). For higher trap stiffness, the main response was an increase in vinculin recruitment, while the tension did not increase significantly. The increase in vinculin intensity was correlated with the decrease in FRET efficiency at 0.26 pN/nm but not at lower stiffness. Thus, the presence of the high stiffness optical trap over 5 min appears to induce a positive correlation between vinculin recruitment and vinculin tension. In a few instances, vinculin puncta migrated a few microns away from the bead exceeding the bead’s movement speed while experiencing an increase in both vinculin intensity and tension. Taken together, the results suggest that combining an optical trap with vinculin tension measurements uncovers novel vinculin dynamics in the presence of a force. Statement of significance A calibrated system combining optical tweezers with FRET microscopy is used to measure vinculin tension in conjunction with its recruitment in response to a force. The results demonstrate an increase in vinculin recruitment, correlated with stiffness rather than force, but less increase in vinculin tension. At higher stiffness (0.26pN/nm), a correlation between vinculin recruitment and tension is observed but not at lower trap stiffness (0.13 pN/nm) or with no optical trap. Such dynamic measurements, enabled by the techniques presented in this paper, can help elucidate the mechanisms by which cells sense physical forces and the properties of proteins, such as vinculin, which play a fundamental role in cellular behaviors involving tissue growth and repair.

N-Cadherin based adhesion and Rac1 activity regulate tension polarization in the actin cortex

Scientific Reports · 2025-02-04 · 12 citations

Tension-adhesion interplay is a crucial mechanism in multicellular organisms that determines the tension differential among internal and external interfaces, which in turn, mediates tissue surface tension and cell sorting, morphogenesis and remodeling, and cancer progression. Cadherins are widely believed to be involved, yet key aspects of the process are neither well characterized nor quantified. This study demonstrates the critical role of N-cadherin in driving tension polarization throughout the actin cortical network. N-cadherin regulates both tension increase at the cell-medium (external) interface and decrease at the cell-cell (internal) interface, and their quantitative magnitudes, both absolute and relative, strongly depend on the surface density of N-cadherin. Furthermore, the strength of tension polarization also increases with respect to the number of cell-cell interfaces for cells within a multicellular cluster. The cadherin-actin contractility linkage is mediated by Rac1, which serves as a molecular switch to trigger cortex remodeling and contraction via myosin II. Inhibition of Rac1 activity decreases tension polarization and leads to reduced coherence in both small clusters and spheroids. These results provide a pathway to reconcile opposing theories for tissue surface tension generation and perspectives in cancer treatment.

Bispecific immune cell engager enhances the anticancer activity of CD16+ NK cells and macrophages in vitro, and eliminates cancer metastasis in NK humanized NOG mice

Journal for ImmunoTherapy of Cancer · 2024 · 18 citations

• Cancer research
• Immunology
• Medicine

BACKGROUND: In a prior report, we detailed the isolation and engineering of a bispecific killer cell engager, referred to as BiKE:E5C1. The BiKE:E5C1 exhibits high affinity/specificity for the CD16a activating receptor on natural killer (NK) cells and human epidermal growth factor receptor 2 (HER2) on cancer cells. In vitro studies have demonstrated that BiKE:E5C1 can activate the NK cells and induce the killing of HER2+ ovarian and breast cancer cells, surpassing the performance of the best-in-class monoclonal antibody, Trazimera (trastuzumab). To advance this BiKE technology toward clinical application, the objective of this research was to demonstrate the ability of BiKE:E5C1 to activate CD16+ immune cells such as NK cells and macrophages to kill cancer cells, and eradicate metastatic HER2+ tumors in NK humanized NOG mice. METHODS: We assessed BiKE:E5C1's potential to activate CD16-expressing peripheral blood (PB)-NK cells, laNK92 cells, and THP-1-CD16A monocyte-macrophages through flowcytometry and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC) assays. Subsequently, laNK92 cells were selected as effector cells and genetically modified to express the nanoluciferase gene, enabling the monitoring of their viability in NK humanized NOG mice using quantitative bioluminescent imaging (qBLI). To evaluate the functionality of BiKE:E5C1 in vivo, we introduced firefly luciferase-expressing ovarian cancer cells via intraperitoneal injection into hIL-15 and hIL-2 NOG mice, creating a model of ovarian cancer metastasis. Once tumor establishment was confirmed, we treated the mice with laNK92 cells plus BiKE:E5C1 and the response to therapy was assessed using qBLI. RESULTS: Fc region had no impact on BiKE:E5C1's anticancer activity. In vivo results reveal that both hIL-15 and hIL-2 NOG mouse models support the viability and proliferation of laNK92 cells. Furthermore, it was observed that BiKE:E5C1 activates laNK92 cells in mice, leading to eradication of cancer metastasis in both NK humanized hIL-15 and hIL-2 NOG mouse models. CONCLUSIONS: Collectively, our in vivo findings underscore BiKE:E5C1's potential as an immune cell engager capable of activating immune cells for cancer cell elimination, thereby expanding the arsenal of available BiKEs for cancer immunotherapy.

Self-Assembled Fibroblast Growth Factor Nanoparticles as a Therapeutic for Oxidant-Induced Neuronal and Skin Cell Injury

ACS Applied Bio Materials · 2024-07-22 · 7 citations

Traumatic brain injury (TBI) and spinal cord injury (SCI) are neurological conditions that result from immediate mechanical injury, as well as delayed injury caused by local inflammation. Furthermore, TBI and SCI often lead to secondary complications, including pressure wounds of the skin, which can heal slowly and are prone to infection. Pressure wounds are localized areas of damaged tissue caused by prolonged pressure on the skin due to immobility and loss of neurological sensation. With the aim to ameliorate these symptoms, we investigated whether fibroblast growth factors 2 (FGF-2) could contribute to recovery. FGF-2 plays a significant role in both neurogenesis and skin wound healing. We developed a recombinant fusion protein containing FGF-2 linked to elastin-like polypeptides (FGF-ELP) that spontaneously self-assembles into nanoparticles at around 33 °C. The nanoparticle's size was ranging between 220 and 250 nm in diameter at 2 μM. We tested this construct for its ability to address neuronal and skin cell injuries. Hydrogen peroxide was used to induce oxidant-mediated injury on cultured neuronal cells to mimic the impact of reactive oxidants released during the inflammatory response in vivo. We found that FGF-ELP nanoparticles protected against hydrogen peroxide-mediated injury and promoted neurite outgrowth. In the skin cell models, cells were depleted from serum to mimic the reduced levels of nutrients and growth factors in chronic skin wounds. FGF-ELP increased the proliferation and migration of human keratinocytes, fibroblasts, and endothelial cells. FGF-ELP is, therefore, a potentially useful agent to provide both neuroprotection and promotion of cellular processes involved in skin wound healing.

Comparison of vinculin tension in cellular monolayers and three-dimensional multicellular aggregates

Biomedical Optics Express · 2024-08-06 · 2 citations

Confocal frequency-domain fluorescence lifetime and Förster resonance energy transfer (FRET) microscopy of Chinese hamster ovary (CHO-K1) cells expressing the vinculin tension sensor (VinTS) is used to compare vinculin tension in three-dimensional (3D) multicellular aggregates and 2D cellular monolayers. In both 2D and 3D cultures, the FRET efficiency of VinTS is 5-6% lower than that of VinTL (p < 0.05), a tail-less control which cannot bind actin or paxillin. The difference between VinTS and VinTL FRET efficiency can be mitigated by treatment with the Rho-associated kinase inhibitor Y-27632, demonstrating that VinTS is under tension in both 2D and 3D cultures. However, there is an overall decrease in FRET efficiency of both VinTS and VinTL in the 3D multicellular aggregates compared with the 2D monolayers. Expression of VinTS in 2D and 3D cultures exhibits puncta consistent with cellular adhesions. While paxillin is present at the sites of VinTS expression in the 2D monolayers, it is generally absent from VinTS puncta in the 3D aggregates. The results suggest that VinTS experiences a modified environment in 3D aggregates compared with 2D monolayers and provide a basis for further investigation of molecular tension sensors in 3D tissue models.

The Use of Collagen Methacrylate in Actuating Polyethylene Glycol Diacrylate–Acrylic Acid Scaffolds for Muscle Regeneration

Annals of Biomedical Engineering · 2023 · 6 citations

• Biophysics
• Chemistry
• Biomedical engineering

Cadherin-based adhesion regulates mechanical polarization in the actin cortex through Rac1

Research Square · 2023-09-15

Re: “Organ-On-A-Chip Technologies for Advanced Blood–Retinal Barrier Models,” by Ragelle et al.

Journal of Ocular Pharmacology and Therapeutics · 2022-06-01

Cell adhesion upregulates the cortical tension and actin cortex thickness during the adhesion process

Biophysical Journal · 2022-02-01

A non-transformed oligodendrocyte precursor cell line, OL-1, facilitates studies of insulin-like growth factor-I signaling during oligodendrocyte development

UNC Libraries · 2020-11-03

The process by which oligodendrocyte progenitors differentiate into mature oligodendrocytes is complex and incompletely understood in part because of the paucity of oligodendrocyte precursors cell lines that can be studied in culture. We have developed a non-immortalized rat oligodendrocyte precursor line, called OL-1, which behaves in a fashion consistent with developing oligodendrocytes in vivo. This OL-1 line provides a model for the study of oligodendrocyte development and offers an alternative to the CG-4 cell line. When OL-1 cells are propagated in conditioned growth media, they have morphology consistent with immature oligodendrocytes and exhibit A2B5 antigen positive and myelin basic protein-negative immunoreactivity. Withdrawal of conditioned growth media and culture in serum-free medium results in OL-1 cell maturation, manifested by a shift to myelin basic protein-positive immunoreactivity, A2B5 antigen-negative immunoreactivity, decreased NG2 mRNA expression, increased expression of proteolipid protein mRNA, and increased expression of CNP protein. In addition, the expression of proteolipid protein and its splicing variant DM-20 exhibit a pattern that is similar to brain proteolipid protein expression during development. When OL-1 cells are exposed to Insulin-like growth factor-I, there are significant increases in proteolipid protein mRNA expression ( p < 0.05), the number of cell processes ( p < 0.05), and cell number ( p < 0.05). Treatment with the caspase inhibitors Z-DEVD-FMK and Z-VAD-FMK (inhibitors of caspases 3, 6, 7, 8, 10 and 1, 3, 4, respectively), Insulin-like growth factor-I, or both, results in a similar increase in cell number. Because Insulin-like growth factor-I does not substantially increase the BrdU labeling of OL-1 cells, these data collectively indicate that Insulin-like growth factor-I increases OL-1 cell number predominately by promoting survival, rather than stimulating proliferation. This non-immortalized oligodendrocyte precursor cell line, therefore, exhibits behavior consistent with the in vivo development of oligodendrocytes and provides an excellent model for the study of developing oligodendrocytes.