DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Development and application of high-speed force spectroscopy for biology – BioHSFS

Development and application of high-speed force spectroscopy for biology

The mechanics of biological systems is crucial for their function. Individual proteins require flexibility of secondary structures to allow microsecond conformational changes. In the living cell, shape maintenance and structural stability is assured by the mechanics of the cytoskeleton, adhesion complexes and the plasma membrane. Thus, the study of the mechanics of proteins, protein complexes and the membrane is important to understand biological processes.

The long-term goal of this project is to develop and apply high-speed force spectroscopy methods to probe the mechanics of biological systems.

The development of nanotechnologies opened the field of single biomolecule mechanics. Atomic force microscopy (AFM) combines force measurements and imaging capabilities with piconewton sensitivity and nanometer resolution. However, the response time in force is still limited to the millisecond timescale, while images are acquired in minutes. The development of high-speed AFM (HS-AFM) using short cantilevers provided ~1000-fold increase in image acquisition rate. We have recently developed high-resolution force mapping allowing submolecular resolution of the mechanical properties of membrane proteins, and high-speed force spectroscopy (HS-FS) that allows force measurements on single proteins at mm/s velocities and µs response time. The long-term goal of this project is to develop and apply high-speed force spectroscopy methods to probe the mechanics of biological systems. The project involves important technological development and addresses relevant biological questions. It is divided in two aims:<br /><br />Aim 1. Application of single molecule high-speed force spectroscopy to probe cell adhesion complexes and protein unfolding<br /><br />Aim 2. Development and application of high-speed force mapping on biological systems

The project involves important technological development and addresses relevant biological questions. It is divided in two aims:

Aim 1. Application of single molecule high-speed force spectroscopy to probe cell adhesion complexes and protein unfolding
We will improve HS-FS to allow automatized and robust measurements of receptor-ligand interactions and multidomain protein unfolding. We will apply the system to probe the binding strength of integrin/ligand bonds and ligand unfolding at physiologically relevant high pulling rates. This application will require the implementation of high sampling rate hardware and software and novel bioconjugation techniques using fusion proteins.

Aim 2. Development and application of high-speed force mapping on biological systems
We will develop high-speed force mapping combining topographic and mechanical imaging at rates 1000 times faster than conventional AFM. To observe how the mechanical properties of individual bacterorhodopsin proteins change dynamically during their photocycle, we will apply high-speed force mapping on purple membranes. As a proof of concept, we will implement high-speed recognition imaging on 2D streptavidin crystals using biotin-coated tips, setting the basis of the technique to the future application on living cells.

1. We have implemented the high-speed force spectroscopy (HS-FS) setup allowing 50 MS/s of sampling rate without memory problems and automatized XY positioning. Although not proposed in the original project, the system has been used to determine the viscoelasticity of living cells. The resulting work has been accepted for publication in Nature Physics. We are now applying it to one of the initial objectives, unfolding the designed chimera protein (point 4 below).
2. We have implemented software written in Matlab for data processing of unbinding and unfolding events that works semi-automatically. It allows automatic detection of relevant force events and determination of the characteristic parameters: loading rate, unbinding/unfolding force, distance to rupture… Part of the software is now being written in python language, allowing even faster accessing to the data.
3. All the necessary components of the position detection system have been purchased and the system is currently being tested on an optical bench for later implementation on the HS-FS system.
4. We have implemented an immobilization strategy that allows covalent coupling of the desired protein via a ybbR tag. The strategy has been successfully tested on a chimera protein designed, expressed and purified for this purpose. The chimera protein includes a ybbR tag, 8 titin I91 domains and a dockerin III receptor. Using cohesin III coated AFM tips, we have been able to improve the frequency of successful events by >50-fold. This provides a more reliable, reproducible and robust method to study protein unfolding. A similar fusion protein is now being built but replacing the titin domains with ICAM-1. A manuscript is being written explaining the method and its application for protein unfolding experiments.

The developed system has been applied to determine the viscoelasticity of living cells at high frequencies, up to 100 kHz. This is a regime that was never explored before using active microrheology. Our results suggest that the viscoelastic response of living cells at high frequencies provides a more univocal fingerprint of the state of the cell and the cytoskeleton. The comparison between benign and malignant cells response suggests the method as a possible diagnosis or prognosis tool in the future. The resulting work has been accepted for publication in Nature Physics. This development, not only represents a remarkable technical advance, but it pushes the system a step forward towards the application of high-speed force measurements to living cells, the long-term goal of this project.

The original project proposed the application of the system to probe adhesion complexes on purified proteins. The demonstrated capacity of working with living cells induces us to modify this original objective, to directly probe them on living cells, thus, in their natural environment.

Revues à comité de lecture
1. Rigato A, A Miyagi, S Scheuring, and F Rico*. High-frequency microrheology reveals cytoskeleton dynamics in living cells. (Accepted in Nature Physics)

Ouvrages ou chapitres d’ouvrage
1. Sumbul F and F Rico. Single-Molecule Force Spectroscopy: Experiments, Analysis And Simualtions. (Submitted to Methods in Molecular Biology)

Communications (conférence)
1. January, 2017 / Single Molecule Biophysics Meeting, Aspen, CO, USA
2. March 2016 / MechanoBiology Meeting, Amsterdam, Netherlands
3. February 2016 / Biophysical Society Meeting, Los Angeles, CA, USA
4. October 2016 / PhysBio 2016, Orsay

The mechanics of biological systems is crucial for their function. Individual proteins require flexibility of secondary structures to allow microsecond conformational changes. In the living cell, shape maintenance and structural stability is assured by the mechanics of the cytoskeleton, adhesion complexes and the plasma membrane. Thus, the study of the mechanics of proteins, protein complexes and the membrane is important to understand biological processes. The development of nanotechnologies opened the field of single biomolecule mechanics. Atomic force microscopy (AFM) combines force measurements and imaging capabilities with piconewton sensitivity and nanometer resolution. However, the response time in force is still limited to the millisecond timescale, while images are acquired in minutes. The development of high-speed AFM (HS-AFM) using short cantilevers provided ~1000-fold increase in image acquisition rate. We have recently developed high-resolution force mapping allowing submolecular resolution of the mechanical properties of membrane proteins, and high-speed force spectroscopy (HS-FS) that allows force measurements on single proteins at mm/s velocities and µs response time. The long-term goal of this project is to develop and apply high-speed force spectroscopy methods to probe the mechanics of biological systems. The project involves important technological development and addresses relevant biological questions. It is divided in two aims:

Aim 1. Application of single molecule high-speed force spectroscopy to probe cell adhesion complexes and protein unfolding
We will improve HS-FS to allow automatized and robust measurements of receptor-ligand interactions and multidomain protein unfolding. We will apply the system to probe the binding strength of integrin/ligand bonds and ligand unfolding at physiologically relevant high pulling rates. This application will require the implementation of high sampling rate hardware and software and novel bioconjugation techniques using fusion proteins.

Aim 2. Development and application of high-speed force mapping on biological systems
We will develop high-speed force mapping combining topographic and mechanical imaging at rates 1000 times faster than conventional AFM. To observe how the mechanical properties of individual bacterorhodopsin proteins change dynamically during their photocycle, we will apply high-speed force mapping on purple membranes. As a proof of concept, we will implement high-speed recognition imaging on 2D streptavidin crystals using biotin-coated tips, setting the basis of the technique to the future application on living cells.

The applicant has experience in force mapping, has developed the first high-speed force spectroscopy system and has already worked with the proposed biological systems, assuring the feasibility of the project. The development and application of high-speed force methods to well-known biological systems will make the technique progress towards the final application: the living cell. The consolidation of HS-FS will allow exploring new dynamic regimes of single biomolecule mechanics. High-speed force mapping opens the door to the study of dynamic changes of the mechanics of proteins, membranes and cells. The expected outcomes are technological and scientifically relevant and may lead to industrial patents. The results will be published in interdisciplinary and specialized journals. Finally, if funded, this project will allow the applicant to establish a new line of research within the hosting lab, complementing it and profiting from its knowledge and expertise in HS-AFM and biological membranes.

Project coordination

Felix RICO (Inserm UMR_S 1006)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

BIO-AFM-LAB Inserm UMR_S 1006

Help of the ANR 261,916 euros
Beginning and duration of the scientific project: September 2015 - 36 Months

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