Mechanics and function of immune cells studied at the single molecule level – ImmuPhy
Mechanics and function of immune cells studied at the single molecule level
B-Lymphocytes are the antibody producing cells of human body are specialized in antigen processing and presentation. Recent experiments showed that upon antigen presentation they undergo an anisotropic distribution of polarity proteins with a consequent cell cytoskeleton rearrangement. The general goal of this research project was to evaluate the contribution of mechanics and cell polarity to the spatio-temporal regulation of antigen acquisition and presentation.
Mechanics and polarity of B cells
We aimed at evaluating the contribution of mechanics to the spatio-temporal regulation of cell polarity in B cells by using an innovative interdisciplinary approach –which combines single molecule experiments in live cells (single protein tracking) and force measurements. Our approach will ultimately be complemented with a theoretical description in terms of active gels with the ultimate goal being to build up a unified physical model that links the mechanical properties of immune cells to their biological function. <br />Our program consisted in performing experiments on single cells using the most advanced imaging techniques. To get a coherent picture of the phenomenon of B cell polarization we needed information on the distribution of the key proteins involved on the long time scale, their behavior on the short time scale, and in response to system perturbations. More specifically we intended to address the three following points:<br />1. Image MyoII, MTOC and lysosomes to understand the spatio temporal organization of the different actors upon Ag presentation;<br />2. Understand the dynamics of a single molecular motor (in particular MyoII molecules) on the short time-scale;<br />3. Correlate images and force measurements using optical tweezers to understand how the force is originated.<br />However some technical difficulties constrained the study to slightly different objects and molecules from the ones initially suggested and in particular we abandoned the observation of Myosin II and the force measurement since they were not possible with the approach suggested in the project.
The main plan of the project was to investigate the mechanics of B lymphocyte polarization following contact with antigen, a crucial step in the antigen recognition and subsequent production of antibodies. We aim at evaluating the contribution of mechanics to the spatio-temporal regulation of cell polarity in B cells by using an innovative interdisciplinary approach which combines single molecule experiments in live cells (single protein tracking) with force measurements and will be further complemented with a theoretical description in terms of active gels. Our ultimate goal is to build up a unified physical model that links the mechanical properties of the B cells to their biological function.
We show that upon contact with a tethered antigen B cell polarize their microtubules organizing center towards the antigen. Simultaneously the formation of a well organised synapse allow the cell to gather the antigen and then internalise it.
We show that the polarization depend on a number of molecules that are relevant for other types of polarity (in tissue, in cell migration etc) such as Cdc42 and Par3. In particular we use single molecule techniques to show how BCR dynamics is influenced by this polarization and how the molecular motors dynein is used by the cell to dock the MTOC.
In conclusion the initial proposal has been only partially fulfilled due to intrinsic difficulties of the system but the ANR funding has allow us to open new research lines and successfully re-orient our plans to more feasible tasks concerning the mechanics of B cells. Moreover the setup and analytical techniques initially proposed were successfully implemented and made available to other projects.
• Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension. (2014) Accepté en Molecular Biology of the Cell
• How B cells capture, process and present antigens: a crucial role for cell polarity. Nat.Rev.Imm. 13(7):475-486 (2013).
• Kinesin KIFC1 actively transports bare double-stranded DNA. Nucl.Acids Res. 41 (9), 4926 (2013).
• Quantum dots to tail single bio-molecules inside living cells. Adv.Drug Del.Rev. 64 (2), 167-178 (2012).
• Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation. Immunity 35, 3, 361–374 (2011).
• Polarity protein Par3 controls B cell receptor dynamics and antigen extraction at the immune synapse. En revision en E-life.
• Taxol effect on kinesin intracellular motion. En preparation
The immune system is constituted by different cell populations, which circulate between lymphoid organs and peripheral tissues as a result of their high migratory capacity. The success of the immune response relies on the ability of these cells to couple their individual immune function with cell motility in response to biochemical and mechanical environmental signals. Cell polarity driven by the contractility of the acto-myosin cytoskeleton has recently emerged as a pivotal regulatory event in the control of the migratory capacity of immune cells as well as in the acquisition of their individual immune function.
We aim at evaluating the contribution of mechanics to the spatio-temporal regulation of cell polarity in immune cells by using an innovative interdisciplinary approach -which combines single molecule experiments in live cells (single particle tracking) and force measurements- and will be further complemented with a theoretical description in terms of active gels. Our ultimate goal is to build up a unified physical model that links the mechanical properties of immune cells to their biological function.
The originality of our project thus lays on the novelty of the question it aims at addressing as well as on the interdisciplinary approach it proposes, which combines physics, cell biology and immunology. The impact of mechanical cues to the spatio-temporal regulation of immune cell functions is highly relevant to their physiology and has not been extensively addressed so far. This project will thus lead to a better understanding of immunity and may, in the long term, help developing novel treatments for immune-related diseases. It will thus contribute to the creation of new bridges between physics and biology and between fundamental and applied sciences.
Project coordination
Paolo PIEROBON (INSTITUT CURIE - SECTION DE RECHERCHE)
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
IC INSTITUT CURIE - SECTION DE RECHERCHE
Help of the ANR 239,200 euros
Beginning and duration of the scientific project:
- 36 Months
