PhD PROPOSAL FOR THE PASTEUR-PARIS UNIVERSITY INTERNATIONAL PROGRAM

 

Deadline for full application: December 15th, 2013

Interviews: March, 2014

Start of the Ph.D.: October 1st, 2014

 

 

Department: Cell Biology and Infections

 

Title of the PhD project: Super-resolution imaging of chromosomes or HIV

 

Name of the lab: Computational Imaging and Modeling Unit

 

Head of the lab: Christophe Zimmer

 

PhD advisor: C. Zimmer + collaborator

 

Email address: czimmer@pasteur.fr

 

Web site address of the lab: https://sites.google.com/site/imagingandmodeling/

 

Doctoral school affiliation and University:

Frontiers in Life Sciences (FdV, Univ Paris 5 + 7) and Interdisciplinary graduate school for life sciences (Iviv, Univ Paris 6)

 

 

Presentation of the laboratory and its research topics:

 

Our lab develops computational and experimental approaches to image and model cellular processes. We combine approaches from informatics, microscopy, physics and cell biology, often in close collaboration with experimental biology groups based at Pasteur. Our current activities concentrate on two areas of research: 1) the spatial organization of the genome and its functional consequences in yeast and other organisms, and 2) the interaction of pathogens, notably HIV, with cellular hosts.

We heavily rely on high resolution and high throughput imaging techniques set up in our lab (particularly PALM/STORM1,2) and on continual development of computational analysis and simulation methods. We also aim to acquire new expertise in experimental biology approaches.

 

Description of the project:

(1 page, Arial font size 11 : 600 words in total with at least 50% dedicated specifically to the proposed PhD project(s))

 

We seek a PhD candidate to work on a project involving super-resolution light microscopy, with applications to nuclear architecture or the early HIV replication cycle. Depending on the candidate's profile and interests, the project could emphasize computational/mathematical work or experimental work, or both.

 

1. Imaging chromosome conformations:

The genome has a complex, non-random organization inside the 3D volume of nuclei, and this organization has important consequences for DNA repair3 and gene expression4. Yet, despite intense current interest, the geography of nuclei and the 3D structures of chromosomes remain largely unknown. The most promising experimental technique, chromosome conformation capture (3C/Hi-C), provides contact frequency maps across the genome, but this technique provides only very indirect and averaged information about chromosome structures5,6. Over the last years, we have contributed to mapping the territorial organization of the yeast nucleus using imaging methods7,8 and have developed a predictive model of chromosome dynamic9,10 that successfully explains most experimental hallmarks of yeast nuclear architecture. Despite these efforts, the exact structures and dynamics of chromosomes in this and other organisms are still unclear. The proposed PhD project (in collaboration with the Darzacq, Bertrand and Koszul labs) aims to fill this gap. The objective is to use high-resolution PALM/STORM imaging techniques available in our lab11,12 to determine the 3D structure and dynamics of chromosomes in single cells, relative to each other and nuclear landmarks. This will be addressed in fixed cells using RNA-FISH and combinatorial labeling approaches that permit discriminating many individual loci (and monitor their expression status). To assess chromosome dynamics, live cell imaging using photoactivatable histones will be coupled to new computational analyses techniques and polymer models. This work should allow detailed analyses of chromosome structures and their correlations with gene expression and/or DNA damage.

 

2. Structure of the HIV pre-integration complex:

In order to replicate, HIV must enter the cell, transit to the nucleus, enter it through nuclear pores, and integrate its DNA (after completion or reverse transcription) into the cellular chromosomes. The pre-integration complex (PIC) is the structure comprising viral DNA that transits through the pores and is competent for integration of the viral DNA into the host genome. Despite the importance of this complex, its composition and structure remain elusive. This is due in part to the challenges of isolating native PICs13 and to the lack of molecular specificity of electron microscopy. The proposed PhD project (in collaboration with the Charneau lab) aims to decipher the structure of the HIV PIC using super-resolution fluorescence microscopy14. This project will benefit from several key assets: (i) our prior experience with applying PALM/STORM to analysis of HIV morphology15, (ii) a technique that recapitulates reverse transcription outside the cell, (iii) availability of multiple fluorescent labeling systems, and (iv) an optical system under final development that should enable 3D imaging with <10 nm lateral resolution, adapted to the size of the PIC as estimated from transmission electron microscopy (~40-60 nm)16. Once the PIC has been successfully imaged in vitro, we will use PALM/STORM to visualize the complex and its maturation from the reverse transcription complex in situ. This project is expected to yield fundamental new insights into critical steps of the early HIV replication cycle and may ultimately help identifying new drug targets.

 

 

 

References:

1.        Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

2.        Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3, 793–795 (2006).

3.        Agmon, N., Liefshitz, B., Zimmer, C., Fabre, E. & Kupiec, M. Effect of nuclear architecture on the efficiency of double-strand break repair. Nature cell biology 15, 694–699 (2013).

4.        Misteli, T. Beyond the sequence: cellular organization of genome function. Cell 128, 787–800 (2007).

5.        Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science (New York, N.Y.) 326, 289–93 (2009).

6.        Rosa, A. & Zimmer, C. Computational models of large-scale genome architecture. International review of cell and molecular biology in press, (2013).

7.        Berger, A. B. et al. High-resolution statistical mapping reveals gene territories in live yeast. Nature Methods 5, 1031–1037 (2008).

8.        Thérizols, P., Duong, T., Dujon, B., Zimmer, C. & Fabre, E. Chromosome arm length and nuclear constraints determine the dynamic relationship of yeast subtelomeres. Proceedings of the National Academy of Sciences 107, 2025 (2010).

9.        Wong, H. et al. A Predictive Computational Model of the Dynamic 3D Interphase Yeast Nucleus. Current biology : CB 22, 1881–90 (2012).

10.      Wong, H., Arbona, J.-M. & Zimmer, C. How to build a yeast nucleus. Nucleus (Austin, Tex.) 4, (2013).

11.      Henriques, R. et al. QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nature Methods 7, 339–340 (2010).

12.      Herbert, S., Soares, H., Zimmer, C. & Henriques, R. Single-Molecule Localization Super-Resolution Microscopy: Deeper and Faster. Microscopy and Microanalysis 18, 1419 (2012).

13.      Farnet, C. M. & Haseltine, W. A. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J. Virol. 65, 1910–1915 (1991).

14.      Müller, B. & Heilemann, M. Shedding new light on viruses: super-resolution microscopy for studying human immunodeficiency virus. Trends in microbiology (2013). doi:10.1016/j.tim.2013.06.010

15.      Lelek, M. et al. Superresolution imaging of HIV in infected cells with FlAsH-PALM. Proceedings of the National Academy of Sciences of the United States of America 109, 8564–9 (2012).

16.      Xu, K., Babcock, H. P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat Methods 9, 185–188 (2012).

 

 

 

 

Keywords:

super-resolution microscopy, HIV, nuclear architecture, computational models, yeast.

 

 

Expected profile of the candidate (optional):

 

We are looking for highly motivated individuals with a strong background in one or more of the following or related fields:

  • physics, biophysics
  • applied mathematics
  • computer science 
  • image or signal processing
  • light microscopy
  • cell biology
  • virology

 

 

 

Contact: Christophe Zimmer (czimmer@pasteur.fr)

 

 

Mis à jour le 16/09/2013