Final Report Summary - BIOMIMETIC-CORTEX (Actin-membrane interaction in biomimetic and living cellular systems studied with a novel, high precision optical method)
Actin-membrane interaction in biomimetic and living cellular systems studied with a novel, high precision optical method.
Introduction:
Every cell in the human body has well defined mechanical properties that are adapted to their function and their environment. These mechanical properties are well controlled by the cell, and originate to a large extend for the cytoskeleton. The cytoskeleton is a network of biopolymers that not only gives mechanical stability but is also the main source of intracellular force generation. An important contribution to the mechanical properties of each cell has the actin cortex, which a dense network of the biopolymer actin tat is located right under the plasma membrane. While it is well established that the actin cortex is a key player for the correct functioning of the whole cell, its mechanical properties are hardly understood. However, for the understanding of the complex cellular processes such as cytokinesis or cell motility the quantitative description of the actin cortex is vital. This understanding has special importance in the context of cancer, where malignant cells become motile, thus forming metastases that eventually cause a fatal progression of the tumour.
Main objectives of the project:
To gain complete understanding of the actin cortex we proposed to reconstitute this structure in a biomimetic liposome, and to study the mechanical properties of this artificial object. The reconstitution has been achieved right at the start of the project, and in a first phase we focused on the construction of the apparatus that was built to measure the mechanics of the biomimetic cortex. This apparatus consists of an optical tweezer, equipped with a high accuracy optical detection method to measure the displacement of any object in the focus of the laser. Operated at very low laser powers this device can measure the fluctuation of the membrane of a cell, or a biomimetic liposome. In the context of the current project, we used the membrane fluctuation of the biomimetic liposomes to extract the mechanical properties of the cytoskeleton-membrane systems. Here we used the fact that the higher the mechanical rigidity of the system, the smaller the fluctuations. In this sense, the main objectives of the project can be described by the following points:
1. determination of the membrane fluctuation with sufficient accuracy;
2. establishing a theoretical model to understand and interpret these measurements;
3. application of the new method to biomimetic vesicles and living cells.
Main results:
During the course of the project, we fully achieved the first two goals, and we partially achieved the third objective. The membrane fluctuation were successfully measured with a spatial accuracy of < 1 nm and temporal accuracy of 10 µs. The resulting data was Fourier transformed to determine the power spectral density (PSD), which was the main resulting data that was analyzed. Using physical concepts of membrane dynamics, we established a theoretical expression of the expected PSD, which was compared to the measurements. This allows extracting the mechanical parameters such as membrane bending rigidity and membrane tension. We successfully tested the method against know literature values, and against controlled condition in which we applied a know membrane tension. The measurements successfully reproduced the expected values. We are currently preparing a manuscript that will cover these findings. We furthermore applied the method on biomimetic liposomes and living cells. While the results on the biomimetic cortex are yet not conclusive, we have advanced considerably on the understanding of the membrane dynamics of living cells (namely red blood cells). Our results on red blood cells show that the membrane mechanics is a complex process that is partially controlled by active processes, and we are pursuing this project even after the end of the fellowship. The preliminary results obtained on the biomimetic actin cortexe.
Introduction:
Every cell in the human body has well defined mechanical properties that are adapted to their function and their environment. These mechanical properties are well controlled by the cell, and originate to a large extend for the cytoskeleton. The cytoskeleton is a network of biopolymers that not only gives mechanical stability but is also the main source of intracellular force generation. An important contribution to the mechanical properties of each cell has the actin cortex, which a dense network of the biopolymer actin tat is located right under the plasma membrane. While it is well established that the actin cortex is a key player for the correct functioning of the whole cell, its mechanical properties are hardly understood. However, for the understanding of the complex cellular processes such as cytokinesis or cell motility the quantitative description of the actin cortex is vital. This understanding has special importance in the context of cancer, where malignant cells become motile, thus forming metastases that eventually cause a fatal progression of the tumour.
Main objectives of the project:
To gain complete understanding of the actin cortex we proposed to reconstitute this structure in a biomimetic liposome, and to study the mechanical properties of this artificial object. The reconstitution has been achieved right at the start of the project, and in a first phase we focused on the construction of the apparatus that was built to measure the mechanics of the biomimetic cortex. This apparatus consists of an optical tweezer, equipped with a high accuracy optical detection method to measure the displacement of any object in the focus of the laser. Operated at very low laser powers this device can measure the fluctuation of the membrane of a cell, or a biomimetic liposome. In the context of the current project, we used the membrane fluctuation of the biomimetic liposomes to extract the mechanical properties of the cytoskeleton-membrane systems. Here we used the fact that the higher the mechanical rigidity of the system, the smaller the fluctuations. In this sense, the main objectives of the project can be described by the following points:
1. determination of the membrane fluctuation with sufficient accuracy;
2. establishing a theoretical model to understand and interpret these measurements;
3. application of the new method to biomimetic vesicles and living cells.
Main results:
During the course of the project, we fully achieved the first two goals, and we partially achieved the third objective. The membrane fluctuation were successfully measured with a spatial accuracy of < 1 nm and temporal accuracy of 10 µs. The resulting data was Fourier transformed to determine the power spectral density (PSD), which was the main resulting data that was analyzed. Using physical concepts of membrane dynamics, we established a theoretical expression of the expected PSD, which was compared to the measurements. This allows extracting the mechanical parameters such as membrane bending rigidity and membrane tension. We successfully tested the method against know literature values, and against controlled condition in which we applied a know membrane tension. The measurements successfully reproduced the expected values. We are currently preparing a manuscript that will cover these findings. We furthermore applied the method on biomimetic liposomes and living cells. While the results on the biomimetic cortex are yet not conclusive, we have advanced considerably on the understanding of the membrane dynamics of living cells (namely red blood cells). Our results on red blood cells show that the membrane mechanics is a complex process that is partially controlled by active processes, and we are pursuing this project even after the end of the fellowship. The preliminary results obtained on the biomimetic actin cortexe.