Abstract

A challenge of modern biology is to be able to observe exactly where and when molecular machines assemble and act within living cells. This requires techniques that avoid ensemble averaging. By using slimfield fluorescence microscopy, we can observe single protein molecules with a 3 ms temporal resolution and 5 nm spatial precision within live E. coli. This has allowed us to determine the in vivo stoichiometry and architecture of the replisome and other molecular machines, for example MukBEF that acts in chromosome organization. Sister replisomes track independently along the DNA and do not act as part of a replication factory [Cell 133, 90, 2008]. Each replisome contains 3 molecules of the replicative polymerase and the sliding clamp, and single replicative helicases and clamp loaders [Science 328, 498, 2010]. In addition to being able to observe the molecular machines in vivo, we are exploiting techniques that allow us to perturb these machines by using controlled rapid degradation of specific proteins. In complementary experiments, we exploit in vitro single molecule studies using ‘magnetic tweezers’ and ‘DNA curtains’ to gain insight into the function of proteins that act in chromosome segregation [EMBO J 29 , 1423, 2010].