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 protein molecules. Using the recombinant Escherichia coli beta-galactosidase protein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetramer, single P4K molecules could be localized and tracked in the nuclei of permeabilized 3T3 cells at a spatial accuracy of approximately 30 nm and a time resolution of 18 ms. Our previous photobleaching measurements indicated that P4K had two fractions inside the nucleus, a larger mobile and a smaller immobile fraction. The present study supported this observation but revealed a much larger variety of mobility classes. Thus, a fraction of P4K molecules appeared to be truly immobile while another fraction was mobile but confined 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. However, 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 over extended distances by diffusion. However, intranuclear protein diffusion is severely restricted, most likely by multiple association-dissociation events and/or impermeable obstacles.
Kues
T
T
Institut für Medizinische Physik und Biophysik, Westfälische Wilhelms-Universität, D-48149 Münster, Germany.
Peters
R
R
Kubitscheck
U
U
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism*; D003198:Computer Simulation; D004058:Diffusion; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009010:Monte Carlo Method; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C417561:Alexa 488 hydrazide; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D006834:Hydrazines; D007074:Immunoglobulin G; D009842:Oligopeptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; C402304:SV40 nuclear localization peptide; D012709:Serum Albumin; D001616:beta-Galactosidase