Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.

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.

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are components of the splicing machinery that removes introns from precursor mRNA. Like other splicing factors, U snRNPs are diffusely distributed throughout the nucleus and, in addition, are concentrated in distinct nuclear substructures referred to as speckles. We have examined the intranuclear distribution and mobility of the splicing factor U1 snRNP on a single-molecule level. Isolated U1 snRNPs were fluorescently labeled and incubated with digitoninpermeabilized 3T3 cells in the presence of Xenopus egg extract. By confocal microscopy, U1 snRNPs were found to be imported into nuclei, yielding a speckled intranuclear distribution. Employing a laser video-microscope optimized for high sensitivity and high speed, single U1 snRNPs were visualized and tracked at a spatial precision of 35 nm and a time resolution of 30 ms. The singleparticle data revealed that U1 snRNPs occurred in small clusters that colocalized with speckles. In the clusters, U1 snRNPs resided for a mean decay time of 84 ms before leaving the optical slice in the direction of the optical axis, which corresponded to a mean effective diffusion coefficient of 1 m 2 ͞s. An analysis of the trajectories of single U1 snRNPs revealed that at least three kinetic classes of low, medium, and high mobility were present. Moreover, the mean square displacements of these fractions were virtually independent of time, suggesting arrays of binding sites. The results substantiate the view that nuclear speckles are not rigid structures but highly dynamic domains characterized by a rapid turnover of U1 snRNPs and other splicing factors.T he splicing of precursor mRNA in the nucleus is catalyzed by supramolecular assemblies designated as spliceosomes, which comprise more than 70 different proteins and five uridinerich small nuclear RNAs (snRNA; ref. 1). Most of these proteins and the snRNAs are organized in the Uridine-rich small nuclear ribonucleoproteins (U snRNPs), which are classified as U1, U2, U5, and U4͞U6, according to the snRNAs they contain. The snRNAs U1, U2, U4, and U5 are synthesized in the nucleus with a 5Ј-terminal monomethyl-guanosine (m 7 G)-cap structure, transiently exported into the cytoplasm, where a common set of seven core proteins (Sm proteins) bind to the snRNAs Sm site and form a ribonucleoprotein complex called ''Sm core'' (2). Stable association of all Sm proteins is necessary for hypermethylation of the m 7 G cap to the 2,2,7-trimethyl-guanosine (m 3 G)-cap structure (3, 4). Also, several proteins associate specifically with the individual U snRNPs; in the case of U1, those proteins are 70K, U1-A, and U1-C (5). After cap modification and 3Јend processing of the snRNAs (6), the mature snRNP particles are reimported into the nucleus by import receptors. The nuclear localization signal of U1 snRNPs is complex, with the m 3 G-cap structure representing one important signaling component (7,8). A second component is located at the Sm core but has not been defined precisely yet (9). Recent...

Development of cytokine resistance is an important feature of melanoma cells during tumor progression. To study the mechanisms of interleukin-6 resistance, we examined an interleukin-6 sensitive (WM35) and an interleukin-6 unresponsive cell line (WM9). Interleukin-6 treatment resulted in rapid inhibition of cyclin-dependent kinase 2/cyclin E activity and accumulation of the hypophosphorylated retinoblastoma protein in WM35 but not in WM9 cells. In contrast to previous reports, no differences in the expression of the cyclin-dependent kinase 2 inhibitor p21Cip1/WAF1 upon interleukin-6 treatment were found in both cell lines. Interleukin-6-induced inhibition of cyclin-dependent kinase 2 was also not due to changes in protein expression of cyclin-dependent kinase 2, cyclin E, p27Kip1 and cdc25A, a phosphatase positively regulating cyclin-dependent kinase 2 activity. As it is established that interleukin-6 resistance of WM9 cells is not caused by differential interleukin-6 receptor expression, we studied whether this is due to defective interleukin-6 signaling in which activation of signal transducer and activator of transcription 3 is a critical step. WM9 cells showed reduced tyrosine phosphorylation, DNA binding, and delayed nuclear translocation of signal transducer and activator of transcription 3 as compared with WM35 cells. The kinase upstream of signal transducer and activator of transcription 3, Janus kinase 1, was constitutively tyrosine-phosphorylated in WM9 cells and did not respond to interleukin-6 with increased phosphorylation. As compared with WM35 cells, interleukin-6 treatment of WM9 cells was not paralleled by reduced activity of the mitogen-activated protein kinase kinase-1, which suppresses activation of signal transducer and activator of transcription 3. Our data suggest that resistance of advanced melanoma cells to interleukin-6 is associated with reduced inhibition of cyclin-dependent kinase 2, which appears to be a consequence of a complex alteration in interleukin-6 signal transduction.