Nuclear export, enlightened.

T he physicist Richard Feynman once quipped that the key to solving or making significant progress on a scientific problem was to “find the open channel.” For the cell biological problem of nucleus-tocytoplasm transport, the literal open channels have long been known: the nuclear pore complexes (NPCs). However, in the metaphorical sense as well, recent work has significantly “opened” the NPC channel as regards the biophysics of the engagement and transit of outwardbound cargo. In this issue of PNAS, Siebrasse et al. (1) track single molecules of a native messenger RNA and provide details on the approach of these transcripts to the NPCs, the probability and persistence of engagement, and the channel transit times. This impressive study was enabled by the use of an iconic system for tracking specific transcripts in the nucleus of a living cell, together with a recently developed innovation in the method of microscopy used. Although the parts list and supramolecular organization of the NPC are presently understood as well as or better than any component of the nucleus (2, 3), the details of how RNA–protein complexes (mRNP) are exported have remained relatively ill-defined as biophysics and thermodynamics and as studied in vivo with intact cells (reviewed in refs. 4, 5). The intranuclear movement of RNAs from their transcription sites to the nuclear periphery is diffusion-mediated (reviewed in ref. 6) but the subsequent step(s) at which metabolic energy is required is uncertain. Do the nucleoplasm-facing components of the NPC irreversibly snag potential cargo or is the initial encounter more tentative? When it has been positioned within approximately the first nanometer of the NPC central channel, is export irreversibly committed? Is the outward vector of cargo in the transport channel saltatory, with frequent (or infrequent) pauses, or is it perhaps the sum of outbound and inbound translocations with the number and/or single step size of the former eventually exceeding that of the latter? These and other fundamental questions about nuclear export have remained frustratingly refractive to investigation, as their resolution obviously requires the tracking of individual cargoes. The live cell detection of single fluorescent molecules, or even particles with more than one to more than five copies of labeled components, is severely limited by the relatively low signal-to-noise ratio (SNR) obtained with even the brightest dyes and most sensitive cameras. The goal is to reduce the background autofluorescence and yet introduce sufficient excitation light to activate enough of the molecules under interrogation, ideally sparsely populated to ensure single-molecule (or -particle) spatial resolution. A number of recent advances have yielded significant reductions in the SNR for biological specimens, especially those with a deep z axis. The one used by Siebrasse et al. (1) is termed selective plane illumination microscopy, also known as light sheet fluorescence microscopy (LSFM), the latter term having been adopted recently by most leaders in the microscopy field. LSFM (Fig. 1) combines the speed and sensitivity of wide field detection with the enhanced resolution inherent in a 90° decoupling of the planes of excitation and detection (refs 7–9 and refs. therein), so that only those fluorophores in a narrow z-axis “sheet” are excited, with the relatively low autofluorescence generated in the sheet by this illumination geometry contributing significantly to the enhanced SNR. Before the work of Siebrasse et al. (1), two studies of mRNA export in mammalian cells used mRNAs genetically engineered to contain a tandem array of fluorescent tags (10, 11), together with a superregistration microscopy innovation in the latter (11). In contrast, Siebrasse et al. (1) used a system in which naturally occurring, unmodified mRNAs, including ones transcribed from the well characterized Balbiani ring (BR) 1 and 2 genes in the polytene larval salivary gland chromosomes of the dipteran insect Chironomus tentans, can be followed. The BR1 and BR2 mRNPs rank as among the most well characterized mRNA transcripts and ribonucleoprotein particles (12–14). Siebrasse et al. (1) labeled the BR mRNPs by injecting into the incubated salivary glands a fluorescent version of an RNA-binding protein, hrp36, with which these transcripts are known to assemble. The authors thus emphasized that the mRNA– ribonucleoprotein complexes they studied are modified only by virtue of a single dye molecule (Alexa 647) attached to the hrp36 protein via a hexapeptide linker. To label the NPCs, they coinjected an Alexa 546-tagged version of the nucleoporin-avid protein NTF-2, permitting simultaneous detection of the mRNP (red) and NPCs (yellow). The authors’ system had an image acquisition power (the image integration time was 20 ms at a 50-Hz frame rate) that, together with their due attention to dual-color signal registration, allowed single mRNPs to be visualized as they approached and engaged NPCs. A possible pitfall in these experiments was the possibility that some of the red signal could be free hrp36 protein, not complexed with mRNA. The authors cleverly ruled this out by showing that a mutant of hrp36 that cannot bind RNA has an intranuclear mobility that is so fast as to not contribute to the focal signals observed. In movies of the numerous pretransport and engaged transport events, some mRNPs were seen to display a productive sequence of NPC binding and transport, whereas others were observed to more tentatively Fig. 1. In LSFM, an excitation beam, or “sheet” (height × width × thickness, 20 μm × 20 μm × 2 μm), is delivered within the focal plane by using a cylindrical lens (magnification of 60×; NA, 0.3), and the emitted fluorescence is detected by using an objective oriented perpendicular to the illumination path (magnification of 60×; NA, 1.0). (A) Diagram of the overall microscopy system. (B) Schematic of the illumination and detection paths. CCD, cooled CCD; WD, working distance. Reproduced from ref. 16.