Views: 222 Author: Leah Publish Time: 2025-02-13 Origin: Site
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● Design and Function of Vinculin Tension Sensors
>> Types of Vinculin Tension Sensors
>> Calibration and Quantification
>> Advantages
>> Limitations
● Applications in Biomedical Research
>> Cell Adhesion and Migration
>> Neuronal Mechanotransduction
● Frequently Asked Questions (FAQs)
>> 1. What is a vinculin tension sensor?
>> 2. How does a vinculin tension sensor work?
>> 3. What are the advantages of using vinculin tension sensors?
>> 4. What are the limitations of vinculin tension sensors?
>> 5. In what areas of biomedical research can vinculin tension sensors be applied?
Cells are continuously subjected to a variety of mechanical forces that play a crucial role in regulating cellular behavior and function. These mechanical cues can influence processes such as cell adhesion, migration, differentiation, and proliferation. Understanding how cells sense and respond to these forces is essential for comprehending various biological processes and developing novel therapeutic strategies[2][5]. Vinculin, a ubiquitous cytoskeletal protein, plays a central role in mechanotransduction by linking integrins to the actin cytoskeleton at focal adhesions (FAs). Focal adhesions are dynamic protein complexes that mediate cell-extracellular matrix (ECM) interactions and serve as mechanical anchors, allowing cells to sense and respond to external forces[1][2].
Vinculin tension sensors are powerful tools that allow researchers to directly measure the mechanical forces experienced by vinculin molecules within cells. These sensors are based on Förster resonance energy transfer (FRET), a phenomenon in which energy is transferred between two fluorescent proteins (FPs) when they are in close proximity[1][9]. By inserting a flexible linker between the vinculin head and tail domains and flanking it with FPs, researchers can create a sensor that changes its FRET efficiency in response to tension[1]. When vinculin is under tension, the FPs are pulled apart, reducing FRET efficiency. Conversely, when vinculin is under low tension, the FPs are closer together, resulting in higher FRET efficiency[1].
This article explores the design, function, and applications of vinculin tension sensors in biomedical research. We delve into how these sensors work, their advantages and limitations, and their use in understanding mechanotransduction in various biological contexts. Additionally, we will address frequently asked questions (FAQs) to provide a comprehensive overview of this technology.
Vinculin tension sensors are designed to measure the mechanical tension experienced by vinculin, a key protein in focal adhesions. These sensors typically consist of the following components[1][2]:
- Vinculin Head and Tail Domains: The sensor includes the head and tail domains of the vinculin protein, which are essential for its function in mechanotransduction[1].
- FRET Module: A flexible linker, often an elastic polypeptide, is inserted between the head and tail domains. This linker is flanked by two fluorescent proteins (FPs), a donor and an acceptor, enabling Förster resonance energy transfer (FRET)[1][9].
- Fluorescent Proteins (FPs): Common FPs used include cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), or more advanced pairs like Clover and mRuby2, which provide improved photophysical properties[2].
The vinculin tension sensor operates based on the principle of Förster resonance energy transfer (FRET). FRET is a process in which energy is transferred from a donor fluorophore to an acceptor fluorophore when they are in close proximity. The efficiency of FRET is highly dependent on the distance between the donor and acceptor[1][9].
1. High Tension: When vinculin is under tension, the force applied to the sensor causes the distance between the donor and acceptor FPs to increase. This increased distance reduces the efficiency of FRET, resulting in a lower FRET signal[1].
2. Low Tension: Conversely, when vinculin is under low tension, the donor and acceptor FPs are in closer proximity. This proximity increases the efficiency of FRET, resulting in a higher FRET signal[1].
Several types of vinculin tension sensors have been developed to optimize performance and sensitivity. These include:
- VinTS (Vinculin Tension Sensor): The original design consists of the head and tail domains of vinculin with an elastic FRET module inserted between them[1]. It exhibits low FRET efficiency under high tension and high FRET efficiency under low tension[1].
- VinTL (Vinculin Tailless): This probe is missing the tail domain, making it insensitive to tension and resulting in high FRET efficiency[1]. It serves as a control for tension-independent FRET changes[1].
- opt-VinTS (Optimized Vinculin Tension Sensor): This optimized sensor incorporates a nine-repeat extensible domain ((GGSGGS)9) to enhance mechanical sensitivity, allowing for more precise detection of tension variations across vinculin[2].
To accurately interpret the FRET signal from vinculin tension sensors, calibration and quantification are essential[2].
- In Vitro Calibration: Initial calibrations were performed *in vitro* to establish the relationship between FRET efficiency and applied force. However, *in cellulo* conditions often differ significantly from *in vitro* settings due to variations in ionic strength, pH, and crowding effects[2].
- In Cellulo Calibration: To address the limitations of *in vitro* calibration, researchers have developed *in cellulo* calibration methods. These methods involve measuring the mechanical properties of the unstructured polypeptides used in the extensible domain directly within cells[2].
- Model-Based Calibration: A first-principles model can predict TSMod mechanical sensitivity using *in cellulo* measurements of the mechanical properties of unstructured polypeptides used as the extensible domain. This model helps circumvent the need for *in vitro* calibrations and accounts for environmental factors[2].
- FRET Efficiency Analysis: FRET efficiency is quantified by measuring the ratio of acceptor emission to donor emission. Changes in this ratio indicate changes in tension across vinculin[1][9].
FRET Efficiency=IAcceptor\IDonor
- Normalization: FRET efficiency data is often normalized to account for variations in expression levels and background fluorescence[2].
- Direct Measurement of Molecular Forces: Vinculin tension sensors provide a direct way to measure the mechanical forces experienced by vinculin at focal adhesions[1][2].
- High Sensitivity: Optimized sensors like opt-VinTS offer enhanced sensitivity, allowing for the detection of subtle changes in tension distribution within cells[2].
- Real-Time Monitoring: FRET-based sensors can be used for real-time monitoring of vinculin tension in live cells, providing dynamic information about mechanotransduction processes[1].
- Spatial Resolution: These sensors offer spatial resolution, allowing for the mapping of tension distributions within individual focal adhesions and across the cell[2].
- Versatility: Vinculin tension sensors can be used in various cell types and experimental conditions to study mechanotransduction in different biological contexts[1][9].
- Calibration Challenges: Accurate calibration of FRET signals can be challenging due to variations in cellular environments and sensor properties[2].
- Environmental Sensitivity: FRET efficiency can be influenced by factors such as pH, temperature, and ionic strength, requiring careful control of experimental conditions[2].
- Photobleaching: Prolonged exposure to excitation light can cause photobleaching of the fluorescent proteins, reducing the signal intensity over time[2].
- Sensor Perturbation: Overexpression of the tension sensor can potentially perturb endogenous vinculin function and cellular mechanics[2].
- Data Interpretation: Interpreting FRET data requires careful consideration of potential artifacts and controls to ensure accurate conclusions[2].
Vinculin tension sensors have found numerous applications in biomedical research, providing valuable insights into mechanotransduction in various physiological and pathological processes.
Cell adhesion and migration are fundamental processes in development, wound healing, and cancer metastasis. Vinculin plays a critical role in these processes by mediating cell-ECM interactions at focal adhesions[1][2].
- Studies: Vinculin tension sensors have been used to investigate how mechanical forces regulate cell adhesion and migration on different substrates[1]. Researchers have shown that cells exert higher tension on stiffer substrates, promoting stronger adhesion and faster migration[2].
- Findings: These sensors have also revealed that tension across vinculin is associated with the assembly and enlargement of focal adhesions, while low tension is observed in disassembling or sliding focal adhesions[3].
Mechanical forces play a significant role in cancer development and progression. Tumor cells interact with the surrounding extracellular matrix and stromal cells, generating mechanical forces that influence tumor growth, invasion, and metastasis[1].
- Studies: Vinculin tension sensors have been used to study the interplay between tumor cells and osteocytes (bone cells) in the bone microenvironment[1]. They found that tumor cells exhibit decreased tensile forces and low cellular motility when positioned close to osteocytes[1].
- Findings: These sensors can evaluate the linkage between molecular forces and cellular motility, providing insights into how tumor cells respond to mechanical cues in their microenvironment[1].
Neurons are highly sensitive to mechanical cues, which play a critical role in neuronal development, axon guidance, and synapse formation. Vinculin is essential for mechanotransduction in neurons[9].
- Studies: Vinculin tension sensors have been used in primary cultures of cortical neurons to probe the function of vinculin in neuronal growth cones[9].
- Findings: These studies demonstrate the feasibility of using VinTS to study mechanotransduction in neurons, providing a foundation for understanding how mechanical forces regulate neuronal development and function[9].
Wound healing is a complex process involving cell migration, ECM remodeling, and tissue regeneration. Mechanical forces play a crucial role in regulating these events[2].
- Studies: Vinculin tension sensors can be used to investigate how mechanical forces influence fibroblast migration and ECM deposition during wound healing[2].
- Findings: By measuring tension across vinculin in fibroblasts, researchers can gain insights into how cells respond to mechanical cues from the wound microenvironment and contribute to tissue repair[2].
Mechanical forces are critical in regulating the structure and function of blood vessels. Endothelial cells, which line the inner surface of blood vessels, are constantly exposed to fluid shear stress and mechanical stretch[2].
- Studies: Vinculin tension sensors can be used to study how endothelial cells respond to these mechanical forces, influencing vascular remodeling and disease[2].
- Findings: These sensors can provide insights into the role of vinculin in mediating endothelial cell adhesion, migration, and barrier function under different mechanical conditions[2].
While vinculin tension sensors have significantly advanced our understanding of mechanotransduction, there are still opportunities for further development and application.
- Improved Sensor Design: Future sensors could be designed with enhanced sensitivity, dynamic range, and stability to provide more accurate and reliable measurements of molecular forces[2].
- Multi-Sensor Approaches: Combining vinculin tension sensors with other biosensors, such as those measuring force on other adhesion proteins or ECM stiffness, could provide a more comprehensive picture of mechanotransduction processes[2].
- In Vivo Applications: Developing strategies for using vinculin tension sensors *in vivo* would allow for the study of mechanotransduction in more physiologically relevant contexts[2].
- High-Throughput Screening: Adapting vinculin tension sensor technology for high-throughput screening could facilitate the discovery of novel drugs that modulate mechanotransduction pathways[2].
Vinculin tension sensors are valuable tools for directly measuring the mechanical forces experienced by vinculin within cells. These sensors have provided significant insights into mechanotransduction in various biological processes, including cell adhesion, migration, cancer biology, neuronal mechanotransduction, wound healing, and cardiovascular biology[1][2][9]. Despite some limitations, ongoing advancements in sensor design, calibration methods, and applications continue to expand the utility of vinculin tension sensors in biomedical research. By providing a direct readout of molecular forces, these sensors contribute to a deeper understanding of how cells sense and respond to mechanical cues, paving the way for novel therapeutic strategies targeting mechanotransduction pathways.
A vinculin tension sensor is a molecular tool used to measure the mechanical forces experienced by vinculin, a key protein in focal adhesions. It utilizes Förster resonance energy transfer (FRET) to detect changes in tension across the vinculin molecule[1][9].
The sensor consists of the vinculin head and tail domains, with a flexible linker and two fluorescent proteins (FPs) inserted between them. When vinculin is under tension, the FPs are pulled apart, reducing FRET efficiency. Conversely, when tension is low, the FPs are closer, increasing FRET efficiency[1].
Advantages include direct measurement of molecular forces, high sensitivity (especially in optimized versions like opt-VinTS), real-time monitoring in live cells, spatial resolution, and versatility in various cell types and experimental conditions[1][2].
Limitations include calibration challenges, environmental sensitivity, photobleaching of fluorescent proteins, potential perturbation of endogenous vinculin function, and the need for careful data interpretation[2].
Vinculin tension sensors can be applied in areas such as cell adhesion and migration, cancer biology, neuronal mechanotransduction, wound healing, and cardiovascular biology, providing insights into how mechanical forces influence these processes[1][2][9].
[1] https://www.nature.com/articles/s41598-019-42132-x
[2] https://elifesciences.org/articles/33927
[3] https://pmc.ncbi.nlm.nih.gov/articles/PMC2901888/
[4] https://elifesciences.org/articles/33927/figures
[5] https://pubmed.ncbi.nlm.nih.gov/30024378/
[6] https://www.researchgate.net/figure/nculin-BRET-Tension-Sensor-in-Focal-Adhesions-A-Schematic-of-Bioluminescent-Resonance_fig1_353745106
[7] https://pmc.ncbi.nlm.nih.gov/articles/PMC3711198/
[8] https://www.mdpi.com/1422-0067/25/11/6198
[9] https://pmc.ncbi.nlm.nih.gov/articles/PMC9150715/
