Steven F. Lee

A spindle-like apparatus guides bacterial chromosome segregation

Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.

3D structures of individual mammalian genomes studied by single-cell Hi-C

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

Brightening, Blinking, Bluing and Bleaching in the Life of a Quantum Dot: Friend or Foe?

Semiconductor nanocrystals or quantum dots (QDs) are highly photoluminescent materials with unique optical attributes that are being exploited in an ever-increasing array of applications. However, the complex surface chemistry of these finite-sized fluorophores gives rise to a number of photophysical phenomena that can complicate their use in imaging applications. Fluorescence intermittency (FI), photoluminescence enhancement (PLE) and spectral bluing are properties of QD emission that would appear, at first sight, detrimental to quantitative measurement. Fortunately, developments in rational QD synthesis and surface modification are promising to minimize the effects of these fluorescence instabilities, while applications that exploit them are now coming to the fore. We review recent experimental and theoretical studies of FI, PLE and bluing, highlighting the benefits, as well as complications, they bring to key applications.

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe’s diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20–40 nm in 3D) over a large field of view (12 × 12 μm).

Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus

Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study, we investigate the distribution of HU in Caulobacter crescentus using a combination of super-resolution fluorescence imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve super-resolution imaging with a single excitation source. Through quantification by Ripleys K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. The methods presented in this letter should be of broad applicability in the future study of prokaryotic NAPs.

Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects

We describe the corkscrew point spread function (PSF), which can localize objects in three dimensions throughout a 3.2 μm depth of field with nanometer precision. The corkscrew PSF rotates as a function of the axial (z) position of an emitter. Fisher information calculations show that the corkscrew PSF can achieve nanometer localization precision with limited numbers of photons. We demonstrate three-dimensional super-resolution microscopy with the corkscrew PSF by imaging beads on the surface of a triangular polydimethylsiloxane (PDMS) grating. With 99,000 photons detected, the corkscrew PSF achieves a localization precision of 2.7 nm in x, 2.1 nm in y, and 5.7 nm in z.

Improved Super-Resolution Microscopy with Oxazine Fluorophores in Heavy Water**

Advanced fluorescence microscopy techniques includingsingle-molecule and super-resolution imaging require brightand photostable fluorophores that can be selectively attachedto biomolecules. There is therefore an ongoing interest in thedevelopment of improved chromophores for biology, espe-cially ones that absorb and emit in the near-infrared owing tothe reduced cellular autofluorescence and phototoxicity atthese wavelengths.

PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits

The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.

Increasing the Brightness of Cyanine Fluorophores for Single‐Molecule and Superresolution Imaging

In spite of their relatively low fluorescence quantum yield, cyanine dyes such as Cy3, Cy5, or Cy7 are widely used in single-molecule fluorescence applications due to their high extinction coefficients and excellent photon yields. We show that the fluorescence quantum yield and lifetime of red-emitting cyanine dyes can be substantially increased in heavy water (D2 O) compared with water (H2 O). We find that the magnitude of the quantum yield increase in D2 O scales with the emission wavelength, reaching a particularly high value of 2.6-fold for the most red-emitting dye investigated, Cy7. We further demonstrate a higher photon yield in single-molecule superresolution experiments in D2 O compared to H2 O, which leads to an improved localization precision and hence better spatial resolution. This finding is especially beneficial for biological applications of fluorescence microscopy, which are typically carried out in aqueous media and which greatly profit from the red spectral range due to reduced cellular auto-fluorescence.

Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids

The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson’s disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.