The continued goal of this Initiative is to determine the high resolution structures of two majormigration organelles, adhesions and protrusions. Our approach is to combine structural methods that rangefrom correlative light and high resolution electron microscopy to X-ray and NMR. As part of this effort, we aredeveloping a new imaging concept that allows us to capture by light microscopy the spatial-temporal aspects ofmigration in living cells and to visualize the same structures using high resolution electron microscopy - 3Dcorrelative light and electron microscopy ('top down approaches'). High resolution structures of individualproteins and their detailed interactions with binding partners obtained by NMR, X-ray or tomography ('bottomup approaches') can then be computationally docked into the three-dimensional maps of the complexes. In thefirst phase of the Consortium, this paradigm was applied to the structure of the actin network in protrusions,providing 3D structures of Arp2/3 mediated actin branches and the structural basis for Arp2/3 activation. Wealso determined the ultrastructure of the lamellipodia of living cells using our new 3D correlative EM andspeckle microscopy and determined the vinculin binding site on a-actinin in the presence of integrin. A unifiedframework for segmentation and analysis of 3-D data was developed to enable the identification of assembliesin 3-D structures.
Showing the most recent 10 out of 368 publications
