Pre-ribosomal particles from nucleoli to cytoplasm.

The analysis of nucleocytoplasmic transport of proteins and messenger RNA has been the focus of advanced microscopic approaches. Recently, it has been possible to identify and visualize individual pre-ribosomal particles on their way through the nuclear pore complex using both electron and light microscopy. In this review, we focused on the transport of pre-ribosomal particles in the nucleus on their way to and through the pores.

Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP.

The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient.

Expansion Microscopy of Bacillus subtilis.

Expansion microscopy enables super-resolved visualization of specimen without the need of highly sophisticated and expensive optical instruments. Instead, the method is executed with conventional chemicals and lab equipment. Imaging of bacteria is performed using standard fluorescence microscopy.

Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter.

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter.

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time.

Ribosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively.

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier.

Background: Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS).

Methods: To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated.

Observing and tracking single small ribosomal subunits in vivo.

Ribosomes are formed of a small and a large subunit (SSU/LSU), both consisting of rRNA and a plethora of accessory proteins. While biochemical and genetic studies identified most of the involved proteins and deciphered the ribosomal synthesis steps, our knowledge of the molecular dynamics of the different ribosomal subunits and also of the kinetics of their intracellular trafficking is still limited. Adopting a labelling strategy initially used to study mRNA export we were able to fluorescently stain the SSU in vivo.

Kinetics of transport through the nuclear pore complex.

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 10ms and nuclear export of messenger RNA (mRNA) particles takes 50-350ms, 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.

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins.

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery.

Trajectories and single-particle tracking data of intracellular vesicles loaded with either SNAP-Crb3A or SNAP-Crb3B.

Using a combined approach of pulse chase labeling and single-particle tracking of Crb3A or 3B loaded vesicles we collected trajectories of different vesicle population in living podocyte cells and evaluated statistically their different mobility patterns. Differences in their intracellular mobility and in their directed transport correspond well to the role of Crb3A and 3B in renal plasma membrane sorting (Djuric et al., 2016) [1].

The C-terminal domain controls the mobility of Crumbs 3 isoforms.

The physiological function of epithelia depends on an asymmetric distribution of their membrane domains. Polarity proteins play a crucial role for distribution processes, however, little is known about their mobility in epithelial cells. In this study, we analyzed the intracellular and plasma-membrane-associated mobility of fluorescence-labeled Crb3A and Crb3B.

Transcription regulation during stable elongation by a reversible halt of RNA polymerase II.

Regulation of RNA polymerase II (RNAPII) during transcription is essential for controlling gene expression. Here we report that the transcriptional activity of RNAPII at the Balbiani ring 2.1 gene could be halted during stable elongation in salivary gland cells of Chironomus tentans larvae for extended time periods in a regulated manner.

Nuclear trafficking and export of single, native mRNPs in Chironomus tentans salivary gland cells.

Real-time observation of single molecules or biological nanoparticles with high spatial resolution in living cells provides detailed insights into the dynamics of cellular processes. The salivary gland cells of Chironomus tentans are a well-established model system to study the processing of RNA and the formation and fate of messenger ribonucleoprotein particles (mRNPs). For a long time, challenging imaging conditions limited the access to this system for in vivo fluorescence microscopy.

A single molecule view on Dbp5 and mRNA at the nuclear pore.

Numerous molecular details of intracellular mRNA processing have been revealed in recent years. However, the export process of single native mRNA molecules, the actual translocation through the nuclear pore complex (NPC), could not yet be examined in vivo. The problem is observing mRNA molecules without interfering with their native behavior.

Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy.

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.

Activated STAT1 transcription factors conduct distinct saltatory movements in the cell nucleus.

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.

Balbiani ring mRNPs diffuse through and bind to clusters of large intranuclear molecular structures.

A detailed conception of intranuclear messenger ribonucleoprotein particle (mRNP) dynamics is required for the understanding of mRNP processing and gene expression outcome. We used complementary state-of-the-art fluorescence techniques to quantify native mRNP mobility at the single particle level in living salivary gland cell nuclei. Molecular beacons and fluorescent oligonucleotides were used to specifically label BR2.

Light sheet microscopy for single molecule tracking in living tissue.

Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample.

Cell-penetrating HIV1 TAT peptides can generate pores in model membranes.

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 microM into the GUVs could be observed.

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin.

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.

Single molecule tracking for studying nucleocytoplasmic transport and intranuclear dynamics.

Microscopic imaging of single fluorescent molecules within cells provides a molecular, real-time view of physiological processes in vivo. Single fluorescent molecules produce diffraction-limited light spots in the image plane, which can be localised with a very high precision. In single-molecule fluorescence microscopy (SMF) the achievable localisation precision depends only on the signal-to-noise ratio (SNR) and the stability of the optical setup.

High-contrast single-particle tracking by selective focal plane illumination microscopy.

Wide-field single molecule microscopy is a versatile tool for analyzing dynamics and molecular interactions in biological systems. In extended three-dimensional systems, however, the method suffers from intrinsic out-of-focus fluorescence. We constructed a high-resolution selective plane illumination microscope (SPIM) to efficiently solve this problem.

Imaging and tracking of single hyaluronan molecules diffusing in solution.

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.

Nuclear transport of single molecules: dwell times at the nuclear pore complex.

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.