The dynamic assembly of proteins must be spatially and temporally coordinated for essential processes to occur, from endo- and exocytosis, to cell division and organelle maintenance. Despite the importance of these processes, our physical paradigm for how proteins form mesoscale assemblies is far from complete.

This is in part a consequence of technical limitations. While the organization and dynamics of membrane proteins are heterogeneous, commonly used fluorescence-based measurements lack information at the molecular scale. In contrast, single molecule measurements limited to looking at only a few molecules in a given cell lack ensemble information. Thus, the study of protein assembly has been limited by a lack of spatially resolved, dynamic information on ensembles of molecules. To overcome these obstacles, we develop and use automated super-resolution fluorescence imaging techniques combined with live cell imaging and single molecule tracking to determine how the dynamics of protein assembly are coordinated.


      Nanoscale architecture of the bacterial Z-ring

The bacterial cytoskeletal protein FtsZ, which forms a constricting “Z-ring” during cell division, is the major cytoskeletal protein involved in cell division in almost all prokaryotes, and is a key next-generation antibiotic target. However, the small size of the Z-ring, about 500 nm in diameter at its largest, makes it difficult to observe in vivo. To address this, we created an automated modality of super-resolution fluorescence microscopy, allowing 3D high throughput live cell microscopy at nanoscale resolution. This allows us to reveal a quantitative nanoscale picture of Z-ring organization in live cells; these results improve our understanding of the structural and possible force generating roles of FtsZ in bacterial cell division. We are now applying this new methodology in both prokaryotic and eukaryotic systems. 


 Mitochondrial dynamics


Super-resolution microscopy has the potential to provide a refined structure of different cellular compartments: endoplasmic reticulum (ER), mitochondria, Golgi, and more. These organelles are highly dynamic, reflecting the continuous processes occurring in living cells. Interactions between compartments are involved in intracellular transport, signalling, and cell differentiation and death. We focus on the biophysics of mitochondrial fission and fusion. Using a combined SIM-STORM approach we’re building a system for the multicolor, live-cell superresolution study of mitochondrial dynamics. 




Super-resolution imaging of DNA


We have shown that single molecule imaging can be performed within the nuclei of mammalian cells, with bright, synthetic dyes. By using site-specific dyes such as PicoGreen, we can stain DNA in living cells. We can also label specific genes using FISH. Alternatively, we can intracellularly target specific proteins using specific labeling of genetically encoded tags such as SNAP-, CLIP-, and Halo. Under optimized imaging conditions, single molecule photoswitching allows us to reconstruct superresolution images or perform high-density single molecule tracking. We are now applying these methods to study the transcription factor search and binding processes that underlie gene expression, and the functional organization of chromatin.