Visualization and tracking of single protein molecules in the cell nucleus

A recently developed laser fluorescence videomicroscopy method was used to determine for the first time the intranuclear trajectories of single protei n molecules. Using the recombinant Escherichia coli beta -galactosidase pro tein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetrame r, single P4K molecules could be localized and tracked in the nuclei of per meabilized 3T3 cells at a spatial accuracy of similar to 30 nm and a time r esolution of 18 ms. Our previous photobleaching measurements indicated that P4K had two fractions inside the nucleus, a larger mobile and a smaller im mobile fraction. The present study supported this observation but revealed a much larger variety of mobility classes. Thus, a fraction of P4K molecule s appeared to be truly immobile while another fraction was mobile but confi ned to very small areas. In addition, a large fraction of the P4K molecules appeared to be mobile and to move over extended distances by diffusion. Ho wever, a quantitative analysis showed that at least two subpopulations were present differing widely in diffusion coefficients. Importantly, both the diffusion coefficients and the fractions of these subpopulations were time- dependent. Our results suggest that proteins can move inside the nucleus ov er extended distances by diffusion. However, intranuclear protein diffusion is severely restricted. most likely by multiple association-dissociation e vents and/or impermeable obstacles.