Three-dimensional single particle tracking in a light sheet microscope

Technical development in microscopy, and particularly in fluorescence microscopy, has facilitated the investigation of ever smaller details in biological specimen. The combination of specific labeling of molecular compounds, sophisticated optical setups and sensitive detectors enables observation of single molecules. Using fast video microscopy, it is now possible to directly observe the cell’s molecular machinery at work by tracking single molecules with high spatial and temporal resolution. Single molecule tracking can reveal detailed information about the dynamics of biological processes. However, technical requirements for single molecule detection limit the depth of field to less than 1 μm. Thus, single molecule tracking is typically limited to studying phenomena in planar membranes or, in extended specimen, often relies on two dimensional projections of short trajectory fragments. The work presented here strives to overcome these limitations by combining real-time three-dimensional localization of single particles with an active feedback loop to keep a particle of interest within the observation volume. To this end, a light sheet microscopy setup was designed and assembled around a commercial microscope body. It was equipped with a fast piezo stage for axial sample positioning. Three-dimensional spatial information was encoded in the shape of the point spread function by astigmatic detection and retrieved by real-time image analysis code developed for this purpose. A novel localization metric based on cross-correlation template matching was devised to enable tracking based on a low number of photons detected per particle. During post-processing, relative axial localizations determined from the image data were combined with the piezo stage position to obtain full three-dimensional particle trajectories. Mechanical and optical properties of the setup were thoroughly characterized using appropriate test samples. A temporal resolution down to 1,12 ms was achieved. The localization precision of the method was experimentally determined by repeated imaging of immobilized fluorescent beads. The capability to track single emitters was validated in a biochemical model system. Lipids labeled with a synthetic dye molecule were incorporated in the bilayer membrane of giant unilamellar vesicles and tracked on their spherical surface. Trajectories of more than 20 s duration could be obtained at as little as 130 photons detected per frame. An analysis of the photophysical properties revealed that observation times per particle were limited not by failure of the tracking algorithm but by photobleaching. Applicability of the method in biological specimen was proved by tracking fluorescent nanoparticles micro-injected into C. tentans salivary gland cell nuclei for more than 270 s in several thousand frames. Subsequently, the method was applied to track mRNA and rRNA particles in C. tentans salivary gland cell nuclei. Biomolecules were specifically labeled by complementary oligonucleotides carrying up to three synthetic dye molecules. It was possible to routinely acquire trajectories of particles with a diffusion coefficient of D = 1-2 μm2/s spanning ≥ 4 s and 4-5 μm in axial direction. The longest trajectories lasted more than 16 s and covered 10 μm axially. Both, observation time and axial range, were increased by more than one order of magnitude as compared to standard 2D tracking experiments. It was thus possible to investigate mobility states not on the basis of an ensemble of short observations but for individual particles

The Lantibiotic Nisin Induces Lipid II Aggregation, Causing Membrane Instability and Vesicle Budding

AbstractThe antimicrobial peptide nisin exerts its activity by a unique dual mechanism. It permeates the cell membranes of Gram-positive bacteria by binding to the cell wall precursor Lipid II and inhibits cell wall synthesis. Binding of nisin to Lipid II induces the formation of large nisin-Lipid II aggregates in the membrane of bacteria as well as in Lipid II-doped model membranes. Mechanistic details of the aggregation process and its impact on membrane permeation are still unresolved. In our experiments, we found that fluorescently labeled nisin bound very inhomogeneously to bacterial membranes as a consequence of the strong aggregation due to Lipid II binding. A correlation between cell membrane damage and nisin aggregation was observed in vivo. To further investigate the aggregation process of Lipid II and nisin, we assessed its dynamics by single-molecule microscopy of fluorescently labeled Lipid II molecules in giant unilamellar vesicles using light-sheet illumination. We observed a continuous reduction of Lipid II mobility due to a steady growth of nisin-Lipid II aggregates as a function of time and nisin concentration. From the measured diffusion constants of Lipid II, we estimated that the largest aggregates contained tens of thousands of Lipid II molecules. Furthermore, we observed that the formation of large nisin-Lipid II aggregates induced vesicle budding in giant unilamellar vesicles. Thus, we propose a membrane permeation mechanism that is dependent on the continuous growth of nisin-Lipid II aggregation and probably involves curvature effects on the membrane

A first order phase transition mechanism underlies protein aggregation in mammalian cells

The formation of misfolded protein aggregates is a hallmark of neurodegenerative diseases. The aggregate formation process exhibits an initial lag phase when precursor clusters spontaneously assemble. However, most experimental assays are blind to this lag phase. We develop a quantitative assay based on super-resolution imaging in fixed cells and light sheet imaging of living cells to study the early steps of aggregation in mammalian cells. We find that even under normal growth conditions mammalian cells have precursor clusters. The cluster size distribution is precisely that expected for a so-called super-saturated system in first order phase transition. This means there exists a nucleation barrier, and a critical size above which clusters grow and mature. Homeostasis is maintained through a Szilard model entailing the preferential clearance of super-critical clusters. We uncover a role for a putative chaperone (RuvBL) in this disassembly of large clusters. The results indicate early aggregates behave like condensates

Reelin and CXCL12 regulate distinct migratory behaviors during the development of the dopaminergic system

The proper functioning of the dopaminergic system requires the coordinated formation of projections extending from dopaminergic neurons in the substantia nigra (SN), ventral tegmental area (VTA) and retrorubral field to a wide array of forebrain targets including the striatum, nucleus accumbens and prefrontal cortex. The mechanisms controlling the assembly of these distinct dopaminergic cell clusters are not well understood. Here, we have investigated in detail the migratory behavior of dopaminergic neurons giving rise to either the SN or the medial VTA using genetic inducible fate mapping, ultramicroscopy, time-lapse imaging, slice culture and analysis of mouse mutants. We demonstrate that neurons destined for the SN migrate first radially and then tangentially, whereas neurons destined for the medial VTA undergo primarily radial migration. We show that tangentially migrating dopaminergic neurons express the components of the reelin signaling pathway, whereas dopaminergic neurons in their initial, radial migration phase express CXC chemokine receptor 4 (CXCR4), the receptor for the chemokine CXC motif ligand 12 (CXCL12). Perturbation of reelin signaling interferes with the speed and orientation of tangentially, but not radially, migrating dopaminergic neurons and results in severe defects in the formation of the SN. By contrast, CXCR4/CXCL12 signaling modulates the initial migration of dopaminergic neurons. With this study, we provide the first molecular and functional characterization of the distinct migratory pathways taken by dopaminergic neurons destined for SN and VTA, and uncover mechanisms that regulate different migratory behaviors of dopaminergic neurons

Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-β, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator β-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity

Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference

Mediator Forms Clusters with RNA Polymerase II in Live Stem Cells

Transcription in eukaryotic cells is regulated not only by promoters but also enhancer elements. Mediator is the key transcription factor that links enhancers and promoters. Here, we labelled endogenous Mediator using CRISPR/Cas9 gene editing in mouse embryonic stem cells (mESCs). Using live cell super-resolution imaging we observed that Mediator forms transient sub-diffractive clusters in the nucleus of live stem cells. We also observed stable and large Mediator clusters. A rank-ordered size distribution of Mediator clusters was similar to a rank-ordered distribution of Mediator ChIP-seq data. By orthogonal labeling of Pol II we could observe that stable Mediator clusters co-localize with stable Pol II clusters, and the enhancer associated transcription factors Sox2 and Oct3/4 in live mESCs. Using Lattice Light Sheet Microscopy we directly observed Mediator and Pol II clusters at high temporal resolution and followed their 3D dynamics over extended periods of time. Upon incubation with the BRD4 inhibitor JQ1, known to dissolve super-enhancer signatures in ChIP-seq data, stable Mediator clusters disappeared. Based on our data we hypothesize that super-enhancer domains with high Mediator signal observed in ChIP-seq result from interactions of chromatin stretches with the large protein clusters observed in this study. Co-localization of many other key transcription factors, namely Pol II, Sox2, and Oct3/4, suggests that the entities observed here play an important role in the transcriptional program of mESC