A large part of our work uses live imaging to observe the dynamics of cortical and membrane proteins and understand how they generate forces across cells and tissue.
Over the past decade, since the seminal works of Watanabe(1) and Ponti(2, 3), single-molecule and quantitative speckle analysis have emerged as extremely powerful and successful approaches to understand the kinetic details and the underlying biology of actomyosin network dynamics in cells(4), but also to probe the microscopic mechanical properties of the network. These techniques are not restricted to the analysis of Actin and have been used successfully in a variety of other contexts to study mobility, dynamics and biochemical properties of the considered species(5-8).
We use HILO microscopy to visualize the dynamics of cell surface proteins and record the single-molecule dynamics of our proteins of interest(9). We then use image analysis technique, single-particle tracking and mathematical models of surface proteins kinetics to extract protein mobility and turnover.
In the lab, we combine all these approaches technique to quantify spatial and temporal variations in mobility and turnover for a range of surface proteins, including polarity proteins (PAR-1, PAR-6), actin, and actin dynamics regualtors (nucleators, elongators, severing proteins and crosslinkers), the molecular motor myosin, and actomyosin dynamics regualtors.
These tools give us the opportunity to address a variety of questions in a simple, versatile and minimally invasive manner – a technique that can be applied to any of the large and growing collection of transgenic strains expressing GFP-tagged fusion proteins in C. elegans(9).
IBPS - Laboratoire de Biologie du Développement – ERL 1156
CNRS – Université Pierre et Marie Curie – Inserm
9, quai Saint-Bernard – Bât. C – 6ème Et. – Case 24 – 75252 Paris cedex 05 – France
Tel :+33 (0)1 44 27 42 41 – email : francois.robinATupmc.fr