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 ratelimiting 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.Dbp5 ATPase cycle | nucleocytoplasmic transport | single molecule microscopy | translocation time

Steroid receptor drugs have been available for more than half a century, but details of the ligand binding mechanism have remained elusive. We solved X-ray structures of the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at the helix 6-7 region that extends the ligand binding pocket toward the receptor surface. Since none of the endogenous ligands exploit this region, we hypothesized that it constitutes an integral part of the binding event. Extensive all-atom unbiased ligand exit and entrance simulations corroborate a ligand binding pathway that gives the observed structural plasticity a key functional role. Kinetic measurements reveal that the receptor residence time correlates with structural rearrangements observed in both structures and simulations. Ultimately, our findings reveal why nature has conserved the capacity to open up this region, and highlight how differences in the details of the ligand entry process result in differential evolutionary constraints across the steroid receptors.

Three-dimensional (3D) spatial information can be encoded in two-dimensional images of fluorescent nanoparticles by astigmatic imaging. We combined this method with light sheet microscopy for high contrast single particle imaging up to 200 µm deep within living tissue and real-time image analysis to determine 3D particle localizations with nanometer precision and millisecond temporal resolution. Axial information was instantly directed to the sample stage to keep a moving particle within the focal plane in an active feedback loop. We demonstrated 3D tracking of nanoparticles at an unprecedented depth throughout large cell nuclei over several thousand frames and a range of more than 10 µm in each spatial dimension, while simultaneously acquiring optically sectioned wide field images. We conclude that this 3D particle tracking technique employing light sheet microscopy presents a valuable extension to the nanoscopy toolbox.

Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1–3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.

N-Acetylspermine oxidase (APAO) catalyzes the conversion of N-acetylspermine or N-acetylspermidine to spermidine or putrescine, respectively, with concomitant formation of N-acetyl-3-aminopropanal and hydrogen peroxide. Here we present the structure of murine APAO in its oxidized holo form and in complex with substrate. The structures provide a basis for understanding molecular details of substrate interaction in vertebrate APAO, highlighting a key role for an asparagine residue in coordinating the N-acetyl group of the substrate. We applied computational methods to the crystal structures to rationalize previous observations with regard to the substrate charge state. The analysis suggests that APAO features an active site ideally suited for binding of charged polyamines. We also reveal the structure of APAO in complex with the irreversible inhibitor MDL72527. In addition to the covalent adduct, a second MDL72527 molecule is bound in the active site. Binding of MDL72527 is accompanied by altered conformations in the APAO backbone. On the basis of structures of APAO, we discuss the potential for development of specific inhibitors.

Optical biosensors entered target-based small-molecule drug discovery more than two decades ago and have since transformed into a value-adding component in the decision-making process. Here, we briefly highlight the major application areas of optical biosensors and focus on desirable profiles of such platforms in order to ensure their effective use in small molecule drug discovery. Furthermore, we will emphasize current technology-based constraints and discuss experimental strategies to address these limitations as well as provide a view of necessary technology improvements for next generation platforms.

Hyaluronan is an important soluble component of the extracellular matrix of many tissues with well known space-filling, lubricating and signaling functions. As such, hyaluronan can regulate cell adhesion, migration, differentiation and proliferation. Ultrastructural studies showed the existence of fibers and networks of hyaluronan molecules at surfaces, while bulk studies of hyaluronan in solution indicated that the polymer forms random coils. Here, we show that single hyaluronan molecules can be visualized and tracked in three-dimensional samples at room temperature in aqueous buffer. Using a wide-field fluorescence microscope equipped with laser excitation and an sensitive and fast EMCCD camera for fluorescence detection, single FITC-labeled hyaluronan molecules from rooster comb were detected in aqueous solutions. Freely moving hyaluronan-FITC could be tracked over up to 20 images acquired at a frame rate of 98 Hz. Analysis of the trajectories revealed Brownian motion of hyaluronan in tris-buffered saline with an average diffusion coefficient D=3.0+/-0.2 microm(2)/s. These observations confirm the concept that hyaluronan molecules form random coils in solution. The possibility of following the tracks of single hyaluronan molecules in solution facilitates the analysis of processes that lead to the formation of more organized forms of hyaluronan and its interactions with cells with very high spatial and temporal accuracy.

Light sheet microscopy became a powerful tool in life sciences. Often, however, the sheet geometry is fixed, whereas it would be advantageous to adjust the sheet geometry to specimens of different dimensions. Therefore we developed an afocal cylindrical zoom lens system comprising only 5 lenses and a total system length of less than 160 mm. Two movable optical elements were directly coupled, so that the zoom factor could be adjusted from 1x to 6.3x by a single motor. Using two different illumination objectives we achieved a light sheet thickness ranging from 2.4 µm to 36 µm corresponding to lateral fields of 54 µm to 12.3 mm, respectively. Polytene chromosomes of salivary gland cell nuclei of C.tentans larvae were imaged in vivo to demonstrate the advantages in image contrast by imaging with different light sheet dimensions.