Related Experiment Video
Updated: Jan 23, 2026
06:29
Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells
Published on: July 30, 2020
3.5K
Biophysical basis underlying dynamic Lck activation visualized by ZapLck FRET biosensor.
Rongxue Wan1,2,3, Jenny Wu2, Mingxing Ouyang2
1Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China.
Science Advances
|June 22, 2019
Summary
A novel biosensor reveals that Lck kinase is preactivated in T-cells. Phosphorylation at Y394 is crucial for basal Lck activity, but TCR aggregation can activate even unphosphorylated Lck.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Lymphocyte-specific protein tyrosine kinase (Lck) is essential for T-cell receptor (TCR) signaling.
- Understanding Lck regulation is critical for deciphering immune responses.
Purpose of the Study:
- To develop and utilize a Förster resonance energy transfer (FRET) biosensor to visualize Lck kinase activity in live cells.
- To investigate the role of Lck phosphorylation at Y394 in its basal activity and activation dynamics.
Main Methods:
- Development of a sensitive FRET biosensor, ZapLck, for high spatiotemporal resolution imaging of Lck activity.
- Utilizing Lck-deficient T-cells (JCam) for reconstitution experiments with wild-type Lck (LckWT) and mutant LckY394F.
- Analyzing Lck-Lck interactions and diffusion rates using live-cell imaging.
Main Results:
- ZapLck demonstrated that 62% of Lck signal is preactivated in T-cells.
- Lck preactivation was abolished in Lck-deficient cells and restored by LckWT but not LckY394F.
- LckWT exhibited stronger basal Lck-Lck interaction and slower diffusion than LckY394F.
- TCR aggregation induced strong activation of LckY394F, comparable to LckWT, with similar increases in Lck-Lck interaction and reduced diffusion.
Conclusions:
- Phosphorylation at Y394 is necessary for Lck's basal interaction and preactivation.
- Antibody-induced TCR aggregation can overcome the requirement for Y394 phosphorylation for full Lck activation.
- These findings provide new insights into Lck kinase regulation during TCR signaling.
Related Concept Videos
02:20Dynamic Equilibrium
62.0K
A reversible chemical reaction represents a chemical process that proceeds in both forward (left to right) and reverse (right to left) directions. When the rates of the forward and reverse reactions are equal, the concentrations of the reactant and product species remain constant over time and the system is at equilibrium. A special double arrow is used to emphasize the reversible nature of the reaction. The relative concentrations of reactants and products in equilibrium systems vary greatly;...
62.0K
02:04Co-activators and Co-repressors
8.4K
Gene transcription is regulated by the synergistic action of several proteins that form a complex at a gene regulatory site. This is observed in eukaryotes, where the regulation of gene expression is a complex process. Regulatory proteins in eukaryotes can broadly be classified into two types – regulators that bind directly to specific DNA sequences and co-regulators that associate with regulatory proteins but cannot directly bind to the DNA. These co-regulators are further divided into...
8.4K
02:26tRNA Activation
22.7K
Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
22.7K
01:26Activation Energy
86.4K
Activation energy is the minimum amount of energy necessary for a chemical reaction to move forward. The higher the activation energy, the slower the rate of the reaction. However, adding heat to the reaction will increase the rate, since it causes molecules to move faster and increase the likelihood that molecules will collide. The collision and breaking of bonds represents the uphill phase of a reaction and generates the transition state. The transition state is an unstable high-energy state...
86.4K
01:26Visual System
1.7K
Light enters the eye through the cornea, a transparent, dome-shaped surface covering the surface of the eyeball that helps to direct and focus incoming light. This light is then channeled toward the pupil, an adjustable opening whose size is controlled by the iris. The iris, a pigmented muscle, regulates the amount of light entering the eye by contracting or dilating the pupil, thereby ensuring optimal light levels for clear vision.
Once through the pupil, the light passes through the lens, a...
Once through the pupil, the light passes through the lens, a...
1.7K
01:08Equation of Rotational Dynamics
14.7K
Angular variables are introduced in rotational dynamics. Comparing the definitions of angular variables with the definitions of linear kinematic variables, it is seen that there is a mapping of the linear variables to the rotational ones. Linear displacement, velocity, and acceleration have their equivalents in rotational motion, which are angular displacement, angular velocity, and angular acceleration. Similar to the rotational variables, a mapping exists from Newton's second law of motion...
14.7K