Single molecule microscopy techniques allow to visualize the translocation of single transport receptors and cargo molecules or particles through nuclear pore complexes. These data indicate that cargo molecule import into the nucleus takes less than 10 ms and nuclear export of messenger RNA (mRNA) particles takes 50–350 ms, up to several seconds for extremely bulky particles. This review summarizes and discusses experimental results on transport of nuclear transport factor 2 (NTF2), importin β and mRNA particles. Putative regulatory functions of importin β for the NPC transport mechanism and the RNA helicase Dbp5 for mRNA export kinetics are discussed.

The transport of messenger RNA-protein particles (mRNPs) has been studied by numerous experimental approaches with quite controverse results. Only a close look into experimental details and recent single molecule experiments using the Balbiani ring mRNPs of the salivary gland cells of Chironomus tentans allowed to resolve the contradictory data. Intranuclear mRNP transport requires some ten seconds, and thus is not rate-limiting for gene expression, since transcription and translation take minutes up to hours for very long genes.

We have developed a new molecular beacon design that requires an additional UV pulse for fluorescence activation. This improves the signal-to-noise ratio tremendously compared to previous approaches and allows for a precise control of the time point and location of RNA labelling.

Nuclear export of mRNA is a key transport process in eukaryotic cells. To investigate it, we labeled native mRNP particles in living Chironomus tentans salivary gland cells with fluorescent hrp36, the hnRNP A1 homolog, and the nuclear envelope by fluorescent NTF2. Using light sheet microscopy, we traced single native mRNA particles across the nuclear envelope. The particles were observed to often probe nuclear pore complexes (NPC) at their nuclear face, and in only 25% of the cases yielded actual export. The complete export process took between 65 ms up to several seconds. A rate-limiting step was observed, which could be assigned to the nuclear basket of the pore and might correspond to a repositioning and unfolding of mRNPs before the actual translocation. Analysis of single fluorescent Dbp5 molecules, the RNA helicase essential for mRNA export, revealed that Dbp5 most often approached the cytoplasmic face of the NPC, and exhibited a binding duration of approximately 55 ms. Our results have allowed a refinement of the current models for mRNA export.

Cell-penetrating peptides like the cationic HIV1 TAT peptide are able to translocate across cell membranes and to carry molecular cargoes into the cellular interior. For most of these peptides, the biophysical mechanism of the membrane translocation is still quite unknown. We analyzed HIV1 TAT peptide binding and mobility within biological model membranes. To this end, we generated neutral and anionic giant unilamellar vesicles (GUVs) containing DPPC, DOPC, and cholesterol and containing DPPC, DOPC, cholesterol, and DPPS (DOPS), respectively. First, we characterized the mobility of fluorescently labeled lipids (TR-DHPE) within liquid-ordered and liquid-disordered lipid phases by single-molecule tracking, yielding a DLO of 0.6 ± 0.05 μm2/s and a DLD of 2.5 ± 0.05 μm2/s, respectively, as a reference. Fluorescently labeled TAT peptides accumulated on neutral GUVs but bound very efficiently to anionic GUVs. Single-molecule tracking revealed that HIV1 TAT peptides move on neutral and anionic GUV surfaces with a DN,TAT of 5.3 ± 0.2 μm2/s and a DA,TAT of 3.3 ± 0.2 μm2/s, respectively. TAT peptide diffusion was faster than fluorescent lipid diffusion, and also independent of the phase state of the membrane. We concluded that TAT peptides are not incorporated into but rather floating on lipid bilayers, but they immerged deeper into the headgroup domain of anionic lipids. The diffusion constants were not dependent on the TAT concentration ranging from 150 pM to 2 μM, indicating that the peptides were not aggregated on the membrane and not forming any “carpet”.

Messenger ribonucleoprotein particles (mRNPs) move randomly within nucleoplasm before they exit from the nucleus. To further understand mRNP trafficking, we have studied the intranuclear movement of a specific mRNP, the BR2 mRNP, in salivary gland cells in Chironomus tentans. Their polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm, which allows us to study mRNP mobility without interference of chromatin. The particles were fluorescently labeled with microinjected oligonucleotides (DNA or RNA) complementary to BR2 mRNA or with the RNA-binding protein hrp36, the C. tentans homologue of hnRNP A1. Using high-speed laser microscopy, we followed the intranuclear trajectories of single mRNPs and characterized their motion within the nucleoplasm. The Balbiani ring (BR) mRNPs moved randomly, but unexpectedly, in a discontinuous manner. When mobile, they diffused with a diffusion coefficient corresponding to their size. Between mobile phases, the mRNPs were slowed down 10-to 250-fold but were never completely immobile. Earlier electron microscopy work has indicated that BR particles can attach to thin nonchromatin fibers, which are sometimes connected to discrete fibrogranular clusters. We propose that the observed discontinuous movement reflects transient interactions between freely diffusing BR particles and these submicroscopic structures.

Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites.

Cell-penetrating peptides like the cationic human immunodeficiency virus-1 trans-acting activator of transcription (TAT) peptide have the capability to traverse cell membranes and to deliver large molecular cargoes into the cellular interior. We used optical sectioning and state-of-the-art single-molecule microscopy to examine the passive membrane permeation of fluorescently labeled TAT peptides across the membranes of giant unilamellar vesicles (GUVs). In GUVs formed by phosphatidylcholine and cholesterol only, no translocation of TAT up to a concentration of 2 μM into the GUVs could be observed. At the same peptide concentration, but with 40 mol % of anionic phosphatidylserine in the membrane, rapid translocation of TAT peptides across the bilayers was detected. Efficient translocation of TAT peptides was observed across GUVs containing 20 mol % of phosphatidylethanolamine, which is known to induce a negative curvature into membranes. We discovered that TAT peptides are not only capable of penetrating membranes directly in a passive manner, but they were also able to form physical pores with sizes in the nanometer range, which could be passed by small dye tracer molecules. Lipid topology and anionic charge of the lipid bilayer are decisive parameters for pore formation.

The activation of STAT transcription factors is a critical determinant of their subcellular distribution and their ability to regulate gene expression. Yet, it is not known how activation affects the behavior of individual STAT molecules in the cytoplasm and nucleus. To investigate this issue, we injected fluorescently labeled STAT1 in living HeLa cells and traced them by single-molecule microscopy. We determined that STAT1 moved stochastically in the cytoplasm and nucleus with very short residence times (<0.03 s) before activation. Upon activation, STAT1 mobility in the cytoplasm decreased ∼2.5-fold, indicating reduced movement of STAT1/importinα/β complexes to the nucleus. In the nucleus, activated STAT1 displayed a distinct saltatory mobility, with residence times of up to 5 s and intermittent diffusive motion. In this manner, activated STAT1 factors can occupy their putative chromatin target sites within ∼2 s. These results provide a better understanding of the timescales on which cellular signaling and regulated gene transcription operate at the single-molecule level.

We have developed a new molecular beacon design that requires an additional UV pulse for fluorescence activation. This improves the signal-to-noise ratio tremendously compared to previous approaches and allows for a precise control of the time point and location of RNA labelling.