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ERC

PALMASSEMBLY Report Summary

Project ID: 243016
Funded under: FP7-IDEAS-ERC
Country: Switzerland

Final Report Summary - PALMASSEMBLY (Protein assembly: From the molecular scale to the mesoscale with super-resolution imaging)

The primary goal of this project was to use experiments to inform quantitative models for the biophysics of protein assemblies, in the cellular environment.

Why proteins?
Proteins are a major class of biomolecules, which serve as the primary actors in living systems. They guide important processes such as cell division, telling the cell when to divide, how fast to do it, and with what geometry. Many diseases are associated with defects in protein function in our cells. Likewise, some next-generation antibiotics target proteins to kill bacteria.

How do we study them?
Microscopy is a powerful tool for studying the dynamics and organization of living systems. Super-resolution microscopy allows us to look at much smaller things than was previously possible, even down to the level of single proteins. We want to apply these methods to study protein assemblies. Toward this goal, we have developed several novel super-resolution fluorescence imaging technologies. In particular, we have enabled automated three-dimensional photoactivated localization microscopy (3D PALM) acquisition, developed algorithms for rapid and quantitative analysis, and identified better dyes and buffers for robust multicolor imaging. These advances addressed three bottlenecks in obtaining statistically significant datasets of super-resolution images of biological samples: highly manual low-throughput data acquisition and analysis, lack of quantitative tools for pointillist PALM data, and irreproducible or low quality of multicolor imaging.

What did we learn?
With these technological advances, we have made several exciting findings. In particular, we used automated 3D PALM imaging of hundreds of synchronized bacteria to collect information during different stages of the cell cycle, for hundreds of cells. We used this to study the protein FtsZ, important for directing bacterial cell division. We observed that FtsZ predominantly localizes as a patchy mid-cell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization where FtsZ protofilaments are randomly distributed within the band and interact only weakly.

Centrioles help to guide cell division in eukaryotes like ourselves, so that they divide into just two cells, each with complete genetic information. We optimized chemical buffers to increase resolution by nearly a factor of two, and applied this to study the organization of the key centriolar protein HsSAS-6. For first time, we resolved its nine-fold symmetry in situ in mammalian cells.

Understanding the hierarchical packaging of the DNA polymer and its associated proteins in the nucleus of a eukaryotic cell is an important problem in modern genomics. The packaging mechanism must condense the long DNA polymer (2 meters in the case of the human genome) to fit within the small nuclear volume while still allowing for regulated gene expression. Changes in the physical compaction of chromatin are inferred from biochemical data, but their relationship to gene activation or silencing has not been directly examined. We also tested models of chromatin compaction by measuring the morphology of the developmental regulatory HoxD clusters in single cells from different tissues in developing mice, shedding light on the architecture of these genes during transcriptional activation.

Contact

Caroline VANDEVYVER, (Coordinator International Funding)
Tel.: +41 21 693 4977
Fax: +41 21 693 5583
E-mail
Record Number: 187113 / Last updated on: 2016-07-20
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