Characterization of brain-cytoplasmic actin proprieties

Objectives

The main aims of this project are to :
1) purify and specifically label brain actin,
2) study its dynamic properties and
3) reconstitute a neurocytoskeleton in the presence of both purified microtubules and actin from brains.

Abstract

The cell cytoskeleton, and actin in particular, regulates major biological functions such as cell differentiation, division and migration. While actin is encoded by 6 different genes in humans, most of the data available to date on this protein comes from actin expressed in muscles. Recent data obtained in the lab suggest that cytoplasmic actin found in the brain cannot coassemble with muscle actin, even though these proteins are over 93% identical. The project of this M2 internship is therefore to explore the specific dynamic properties of brain-cytoplasmic actin and its ability to be regulated by known binding proteins (nucleators, stabilizers, etc.).
In a second step, we will explore the ability of brain-cytoplasmic actin to assemble into organized networks as found in cells, and we will recapitulate in vitro, from purified proteins, a neurocytoskeleton composed of organized actin networks and microtubules. This project will provide the basis for in-depth studies into the role of neuronal regulators on the neuronal cytoskeleton.

Methods

Protein expression (bacteria) and purification (chromatography, affinity, polymerization strategy), protein labelling, SDS-PAGE. Imaging (video-microscopy, TIRF). Spectrofluorimetric method to monitor actin polymerization. Analysis (ImageJ/Fiji, R).

Reference

* Elie E et al. (2015) Tau co-organizes dynamic microtubule and actin networks. Sci Rep 5:9964
* Antkowiak et al. (2019) Sizes of actin networks sharing a common environment are determined by the relative rates of assembly PLoS Biol 17(6): e3000317.
* Bagdadi et al. (2024) Stable GDP-tubulin islands rescue dynamic microtubules. J Cell Biol (2024) 223 (8): e202307074.

Requested domains of expertise

Protein biochemistry, Photonic microscopy, Data analysis, Ability to follow protocols, Ability to write and present results.

Contact

A. Antkowiak (MCF)
Email : %20adrien.antkowiakuniv-grenoble-alpes.fr (adrien[dot]antkowiak[at]univ-grenoble-alpes[dot]fr)
Téléphone : +33 (0)4 56 52 05 65