TITRE Projet : A role for F-actin network in HIV

TITRE Projet : A role for F-actin network in HIV

DEMANDE DE BOURSE AMI 2016
Equipe A
Membrane Domains and Virus Assembly Team
CPBS UMR5236
1919 route de Mende
34293 Montpellier
Maitre de stage : Delphine MURIAUX
Equipe B
Research Center for Neurobiology and Neurophysiology
CNRS-AMU UMR 7286
Axonal Domains Architecture team
Faculté de Médecine Secteur Nord
Aix-Marseille Université
Boulevard Pierre Dramard
13344 Marseille Cedex 15
Maitre de stage: Christophe Leterrier
TITRE Projet : A role for F-actin network in HIV-1 Gag assembly as shown by dual color
PALM/STORM microscopy.
SUJET M2 (pour Octobre)
During HIV-1 assembly, the viral Gag proteins are targeted and assembled at the inner leaflet
of the host cell plasma membrane. Gag interacts with specific membrane phospholipids1,
such as PI(4,5)P2 that also participates to the regulation of underneath cortical actin
cytoskeleton dynamics. In turn, the actin network can promote localized membrane
reorganization and could be involved in Gag assembly and particle formation. Our research
team has studied the implication of specific actin effectors on HIV-1 Gag membrane
localization and viral particle release in CD4 T cells, the main HIV-1 targeted cells2. Our
results showed that an activated Rac1 and the IRSp53-Wave2-Arp2/3 signalling pathway are
involved in HIV-1 biogenesis2. Upon HIV-1 Gag expression in T cells, a significant F-actin cell
content increases2. This current work uncovers a new role for the F-actin network in HIV-1
particle formation in CD4 T cells. We now want to characterize, at the cellular and molecular
level, the recruitment of F-actin at HIV-1 Gag assembly and budding sites. For that purpose,
due to the size of a viral budding site (110-140 nm in diameter), we need to study Gag and
the F-actin network with the help of two colors super resolution microscopy. The team A has
already set up the condition for the acquisition of a photoconvertible Gag-mEOS2 proteins in
viral assembly plateforms by sptPALM and PALM/TIRF microscopy imaging in T cells.
Together with the team B, expert in studying intracellular F-actin architectures by STORM3,4,
we will now set up conditions for simultaneously co-imaging Gag-mEOS2 by PALM and label
filamentous-actin (F-actin) by STORM in T cells. We will analyze and quantify coclusterization of identified viral Gag budding spots and F-actin, using ImageJ/Matlab plugins
developed in our 2 teams. We will also characterize F-actin architectures at the vicinity of
the viral budding sites, as compared to cryo-electron tomography5.
All the
biological/virological set up will be performed by team A, the dual color PALM/STORM
optimization by both team, the image acquisition under team B supervision, and the
quantification and result analysis by the teams A and B.
1- Role of Lipids during HIV-1 assembly in T cells and Macrophages. Mariani C., Desdouits M., Favard C., Benaroch P. &
D.Muriaux. (2014) Frontiers in Microbiology, 5:312.
2- Involvement of the Rac1-IRSp53-Wave2-Arp2/3 signalling pathway in HIV-1 Gag particle release in CD4 T cells. Thomas
A, Mariani-Floderer C, López-Huertas MR, Gros N, Hamard-Péron E, Favard C, Ohlmann T, Alcamí J, Muriaux D. J Virol. 2015
Aug;89(16):8162-81.
3- Nanoscale architecture of the axon initial segment reveals an organized and robust scaffold.
Leterrier C.*, Potier J., Caillol G., Rueda Boroni F., Debarnot C. and Dargent B. Cell Reports, 2015 Dec 29;13(12):2781-93.
4- A dynamic formin-dependent deep F-actin network in axons.
Ganguly A., Tang Y., Wang L., Ladt K., Loi J., Dargent B., Leterrier C. and Roy S.
J Cell Biol., 2015 Aug 3;210(3):401-17.
5-The nucleocapsid domain of Gag is dispensable for actin incorporation into HIV-1 and for association of viral budding
sites with cortical F-actin. Stauffer S, Rahman SA, de Marco A, Carlson LA, Glass B, Oberwinkler H, Herold N, Briggs JA,
Müller B, Grünewald K, Kräusslich HG. J Virol. 2014 Jul;88(14):7893-903.