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cytoskeletal dyamics
Currently viewing: 24 September - 4 October 2007 | Gif-sur-Yvette | FR

Our general objective is to provide young researchers in life sciences with a conceptual and experimental tutorial framework enabling them to use in vitro biochemical assays, biomimeic assays and light microscopy physical methods to address major issues regarding the functions of the cytoskeleton in motility and its control by signalling. Within this general objective, specific goals will be pursued.

One part of the course will cover the physical-chemical methods to analyse the dynamics of actin filaments and microtubules and their control by regulatory proteins. Basic assays allowing to quantitatively measure the steady state of actin filament assembly, the kinetics at barbed and pointed ends of actin filaments and how these are affected by regulators will be taught. How the function of a putative actin regulatory protein can be characterised as e.g. G-actin sequestering, barbed end capping, nucleating, profilin-like. using these assays will be explained. The practical course will make the students familiar with fluorescence microscopy techniques and kinetic analysis, derivation of equilibrium and rate constants.

A second part will be devoted to measurements of assembly dynamics of individual polymers (actin filaments and microtubules) and movement of single fluorescently labeled associated proteins, using light microscopy (TIRFM), in particular for the study of processive motors of assembly (formins) or disassembly (kinesins KIF2).

A third part will be devoted to biomimetics and teach how a bottom-up approach aiming at reconstituting an autonomously motile system combining purified essential cellular components in a controlled fashion can be used to understand the mechanism of a motile process generated by site-directed cytoskeleton assembly: several set-ups will be shown like actin-based propulsion of a functionalized solid particle or of a giant liposome; reconstitution of adhesions and associated actin assembly; synergy and co-ordination between two motile modules. Assays will be conducted using phase contrast and single or double fluorescence of labeled molecules (actin, regulatory proteins). Use of the image analysis softwares to obtain quantitative values of crucial parameters will be taught.

More emphasis will be given to the aspects of biology of the cytoskeleton that can be addressed by physics, like force measurements due to growth of single polymers, using optical tweezers, or diffusion of actin nucleating machineries in membranes in connection with interactions with the cytoskeleton using FRAP techniques. The amoeba Dictyostelium discoideum and the fission yeast S. Pombe will be used as genetically tractable animal models for live observations of motile behaviour.

The course is aimed at young scientists/postdocs who plan to develop a project in the field of signalling to motility using in part in vitro mechanistic approaches. We think that understanding biological function of the cytoskeleton requires knowledge of biochemical methods that are usually not covered by cellular and molecular biology academic studies, hence there is a gap to fill here. In addition, most recent discoveries in cell motility have been and are still made by purifying cell components and using reconstitution assays. Moreover, cytoskeleton and motility is a choice field for the interface between physics and biology. Indeed molecular motors and polymer growth develop forces that can be measured using optical tweezers and micro-manipulators. These measurements are made in a relevant and quantifiable fashion only if the biochemical composition of the system is controlled. Hence the mix of biochemistry, biomimetics and physics makes sense within the scope of this course.

In this regard, the course should also attract physicists who are interested in the mechanical properties of the cytoskeleton that support motility and wish to aquire the experimental biological background to design appropriate physical experiments in a biologically relevant context.

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