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. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.

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. This chapter describes a protocol for the expansion microscopy of Bacillus subtilis expressing DivIVA-GFP. In addition, the cell wall was labeled by wheat germ agglutinin. Here, we place emphasis on the challenges of selecting the protein and organism of interest.

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. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.

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. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of ∼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s-1 in vivo.

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 CRB2 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. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2podKO knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants. RESULTS: Crb2podKO mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response. CONCLUSIONS: Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

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. We chose DIM2/PNO1 (Defective In DNA Methylation 2/Partner of NOb1) as labelling target and created a stable cell line carrying an inducible SNAP-DIM2 fusion protein. After bulk labelling with a green fluorescent dye combined with very sparse labelling with a red fluorescent dye the nucleoli and single SSU could be visualized simultaneously in the green and red channel, respectively. We used single molecule microscopy to track single SSU in the nucleolus and nucleoplasm. Resulting trajectory data were analyzed by jump-distance analysis and the variational Bayes single-particle tracking approach. Both methods allowed identifying the number of diffusive states and the corresponding diffusion coefficients. For both nucleoli and nucleoplasm we could identify mobile (D = 2.3-2.8 µm2/s), retarded (D = 0.18-0.31 µm2/s) and immobilized (D = 0.04-0.05 µm2/s) SSU fractions and, as expected, the size of the fractions differed in the two compartments. While the fast mobility fraction matches perfectly the expected nuclear mobility of the SSU (D = 2.45 µm2/s), we were surprised to find a substantial fraction (33%) of immobile SSU in the nucleoplasm, something not observed for inert control molecules.

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 ß and mRNA particles. Putative regulatory functions of importin ß for the NPC transport mechanism and the RNA helicase Dbp5 for mRNA export kinetics are discussed.

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. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.

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. Both variants belong to the Crumbs protein family, which control size and identity of apical membranes in epithelial cells. Fluorescence recovery after photo-bleaching measurements revealed different mobilities for the two Crb3 variants. They also differentially affected mobility and localization of the Pals1/Mpp5 protein, which binds to Crb3A but not to Crb3B. In addition, tracking of intracellular vesicles indicated that Crb3A containing vesicles are slightly more immobile than Crb3B ones. Taken together, our data revealed different intracellular mobility patterns for 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. The transcription halt was triggered by heat shock and affected all RNAPII independently of their position in the gene. During the halt, incomplete transcripts and RNAPII remained at the transcription site, the phosphorylation state of RNAPII was unaltered, and the transcription bubbles remained open. The transcription of halted transcripts was resumed upon relief of the heat shock. The observed mechanism allows cells to interrupt transcription for extended time periods and rapidly reactivate it without the need to reinitiate transcription of the complete gene. Our results suggest a so-far-unknown level of transcriptional control in eukaryotic cells.

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. Recent technical and methodical advantages now allow observing even single molecules in these cells. We describe here the experimental approach and the optical techniques required to analyze intranuclear trafficking and export of single native mRNPs across the nuclear envelope.

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. We used a protein-based labeling approach to visualize single native mRNPs in live salivary gland cells of Chironomus tentans, an iconic system used for decades to study the mRNA life cycle. Recombinant hrp36, the C. tentans homolog of mammalian hnRNP A1, was fluorescence labeled and microinjected into living cells, where it was integrated into nascent mRNPs. Intranuclear trajectories of single mRNPs, including their NPC passage, were observed with high space and time resolution employing a custom-built light sheet fluorescence microscope. We analyzed the kinetics and dynamics of mRNP export and started to study its mechanism and regulation by measuring the turnover-kinetics of single Dbp5 at the NPC.

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. 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.

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. 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/ß 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.

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 BR2.1 mRNPs by an in vivo fluorescence in situ hybridization approach. We characterized two major mobility components of the BR2.1 mRNPs. These components with diffusion coefficients of 0.3 ± 0.02 µm²/s and 0.73 ± 0.03 µm²/s were observed independently of the staining method and measurement technique used. The mobility analysis of inert tracer molecules revealed that the gland cell nuclei contain large molecular nonchromatin structures, which hinder the mobility of large molecules and particles. The mRNPs are not only hindered by these mobility barriers, but in addition also interact presumably with these structures, what further reduces their mobility and effectively leads to the occurrence of the two diffusion coefficients. In addition, we provide evidence that the remarkably high mobility of the large, 50 nm-sized BR2.1 mRNPs was due to the absence of retarding chromatin.

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. 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.

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. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 microm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.

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. Typically values between 20 and 40 nm can be achieved. Highly dynamic processes and Brownian motion characterised by diffusion coefficients <20 microm(2)/sec can be followed by high-speed imaging, hence the method is an ideal tool to study intranuclear protein or ribonucleoprotein particle mobility. In contrast to conventional techniques, different forms of mobility in a heterogeneous system may well be distinguished from each other. Furthermore, specific binding and bimolecular interaction events can be followed at the single molecule level. A prominent example of an application is the study of nucleocytoplasmic transport one molecule at a time. In this case, the high localisation precision allows to analyse the binding site distribution of single molecules at the nuclear pore complex, and the high time resolution allows determination of the binding duration of soluble receptors and transport substrates.

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. 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.