Module 5: Structure and dynamics of viral components
Organisers
D. Muriaux, PE Milhiet, C. Moskalenko
The aim of this module is to provide researchers with a solid background in advanced microscopies suitable for the analysis of both structure and dynamics of viral components during replication, focusing on Single Molecule Localization Microscopy (TIRF/PALM), FRET/FLIM, and Atomic Force Microscopy (AFM).
During viral assembly of enveloped viruses, such as HIV-1 and Influenza, the viral components need to reach and assembled at the inner leaflet of the host cell plasma membrane. Viral proteins can be fused to fluorescent tags and fluorescent viral buds can be monitored by TIRF/PALM on fixed or living cells. Inside the cells, fluorescent viral and cellular protein interactions can be monitored by FRET/FLIM. Released viral particles from these cells can then be characterized by AFM imaging and nanoindentation in order to decipher structural morphology and mechanical properties of the viral particles.
Lectures
The module will contain several plenary and didactic lectures. This will include:
Practical session
Participants will be trained during 3 workshops in TIRF/PALM microscopy, FRET/FLIM and in AFM in order to address retroviral Gag assembling and budding in intact cells as well as the structure and mechanical properties of virus-like particles (VLP). The training also includes data analysis.
- Session 1 - Budding of HIV-1 Gag in intact cells using TIRF/PALM microscopy (by C. Marini and C. Sieben).
- Session 2 – Studying retroviral Gag and cell factor interactions in living cells by FRET/FLIM (H. de Rocquigny and D.Muriaux)
- Session 3 - Morphogenesis of VLP by AFM imaging (by P.E. Milhiet and C. Godefroy) and Nanomechanical properties of VLP by AFM force spectroscopy (by C. Moskalenko).
D. Muriaux, PE Milhiet, C. Moskalenko
The aim of this module is to provide researchers with a solid background in advanced microscopies suitable for the analysis of both structure and dynamics of viral components during replication, focusing on Single Molecule Localization Microscopy (TIRF/PALM), FRET/FLIM, and Atomic Force Microscopy (AFM).
During viral assembly of enveloped viruses, such as HIV-1 and Influenza, the viral components need to reach and assembled at the inner leaflet of the host cell plasma membrane. Viral proteins can be fused to fluorescent tags and fluorescent viral buds can be monitored by TIRF/PALM on fixed or living cells. Inside the cells, fluorescent viral and cellular protein interactions can be monitored by FRET/FLIM. Released viral particles from these cells can then be characterized by AFM imaging and nanoindentation in order to decipher structural morphology and mechanical properties of the viral particles.
Lectures
The module will contain several plenary and didactic lectures. This will include:
- Specific-module lecture 1: C. Moskalenko (ENS Lyon) PE. Milhiet (CBS, Montpellier) Introduction to atomic force microscopy on virus
- Specific-module lecture 2: D. Muriaux, (CPBS Montpellier). Introduction of the biological topic and description of viral sample preparations dedicated to fluorescence imaging and atomic force microscopies
- Specific-module lecture 3: H. de Rocquigny, (LPB, Strasbourg University) FRET/FLIM in fixed and living cells
- Specific-module lecture 4: C.Sieben (EPFL Lausanne) Introduction to single molecule localization microscopy (PALM/STORM)
Practical session
Participants will be trained during 3 workshops in TIRF/PALM microscopy, FRET/FLIM and in AFM in order to address retroviral Gag assembling and budding in intact cells as well as the structure and mechanical properties of virus-like particles (VLP). The training also includes data analysis.
- Session 1 - Budding of HIV-1 Gag in intact cells using TIRF/PALM microscopy (by C. Marini and C. Sieben).
- Session 2 – Studying retroviral Gag and cell factor interactions in living cells by FRET/FLIM (H. de Rocquigny and D.Muriaux)
- Session 3 - Morphogenesis of VLP by AFM imaging (by P.E. Milhiet and C. Godefroy) and Nanomechanical properties of VLP by AFM force spectroscopy (by C. Moskalenko).