Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.

A Familiar Protein-Ligand Interaction Revisited with Multiple Methods.

The interaction of hen egg white lysozyme with the trisaccharide tri-N-acetyl glucosamine has been well-characterized by biophysical methods and structural biology. In this chapter, we present a series of experiments designed to detect and quantify that interaction using several commonly available biophysical methods: thermal shift assay, fluorescence intensity, microscale thermophoresis, isothermal titration calorimetry, and surface plasmon resonance.These experiments have been used for teaching and troubleshooting in a core facility. By taking a set of representative data from several years of practical courses, we are able to demonstrate the robustness of the protocols, calculate confidence intervals for the dissociation constant from each method, and illustrate the degree of consistency between those methods when applied to a simple system in a single location by different experimenters.

Subpopulations of sensorless bacteria drive fitness in fluctuating environments.

Populations of bacteria often undergo a lag in growth when switching conditions. Because growth lags can be large compared to typical doubling times, variations in growth lag are an important but often overlooked component of bacterial fitness in fluctuating environments. We here explore how growth lag variation is determined for the archetypical switch from glucose to lactose as a carbon source in Escherichia coli. First, we show that single-cell lags are bimodally distributed and controlled by a single-molecule trigger. That is, gene expression noise causes the population before the switch to divide into subpopulations with zero and nonzero lac operon expression. While "sensorless" cells with zero preexisting lac expression at the switch have long lags because they are unable to sense the lactose signal, any nonzero lac operon expression suffices to ensure a short lag. Second, we show that the growth lag at the population level depends crucially on the fraction of sensorless cells and that this fraction in turn depends sensitively on the growth condition before the switch. Consequently, even small changes in basal expression can significantly affect the fraction of sensorless cells, thereby population lags and fitness under switching conditions, and may thus be subject to significant natural selection. Indeed, we show that condition-dependent population lags vary across wild E. coli isolates. Since many sensory genes are naturally low expressed in conditions where their inducer is not present, bimodal responses due to subpopulations of sensorless cells may be a general mechanism inducing phenotypic heterogeneity and controlling population lags in switching environments. This mechanism also illustrates how gene expression noise can turn even a simple sensory gene circuit into a bet hedging module and underlines the profound role of gene expression noise in regulatory responses.

Organelle-specific targeting of polymersomes into the cell nucleus.

Organelle-specific nanocarriers (NCs) are highly sought after for delivering therapeutic agents into the cell nucleus. This necessitates nucleocytoplasmic transport (NCT) to bypass nuclear pore complexes (NPCs). However, little is known as to how comparably large NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Here, we developed nuclear localization signal (NLS)-conjugated polymersome nanocarriers (NLS-NCs) and studied the NCT mechanism underlying their selective nuclear uptake. Detailed chemical, biophysical, and cellular analyses show that karyopherin receptors are required to authenticate, bind, and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by regressive staining transmission electron microscopy further resolves the NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei.

Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium.

Transport of membrane and cytosolic proteins in primary cilia is thought to depend on intraflagellar transport (IFT) and diffusion. However, the relative contribution and spatial routes of each transport mechanism are largely unknown. Although challenging to decipher, the details of these routes are essential for our understanding of protein transport in primary cilia, a critically affected process in many genetic diseases. By using a high-speed virtual 3D super-resolution microscopy, we have mapped the 3D spatial locations of transport routes for various cytosolic proteins in the 250-nm-wide shaft of live primary cilia with a spatiotemporal resolution of 2 ms and <16 nm. Our data reveal two spatially distinguishable transport routes for cytosolic proteins: an IFT-dependent path along the axoneme, and a passive-diffusion route in the axonemal lumen that escaped previous studies. While all cytosolic proteins tested primarily utilize the IFT path in the anterograde direction, differences are observed in the retrograde direction where IFT20 only utilizes IFT, and approximately half of KIF17 and one third of α-tubulin utilizes diffusion besides IFT.

Helium Scanning Transmission Ion Microscopy and Electrical Characterization of Glass Nanocapillaries with Reproducible Tip Geometries.

Nanopores fabricated from glass microcapillaries are used in applications ranging from scanning ion conductance microscopy to single-molecule detection. Still, evaluating the nanocapillary tip by a noninvasive means remains challenging. For instance, electron microscopy characterization techniques can charge, heat, and contaminate the glass surface and typically require conductive coatings that influence the final tip geometry. Per contra, electrical characterization by the means of ion current through the capillary lumen provides only indirect geometrical details of the tips. Here, we show that helium scanning transmission ion microscopy provides a nondestructive and precise determination of glass nanocapillary tip geometries. This enables the reproducible fabrication of axially asymmetric blunt, bullet, and hourglass-shaped tips with opening diameters from 20 to 400 nm by laser-assisted pulling. Accordingly, this allows for an evaluation of how tip shape, pore diameter, and opening angle impact ionic current rectification behavior and the translocation of single molecules. Our analysis shows that current drops and translocation dwell times are dominated by the pore diameter and opening angles regardless of nanocapillary tip shape.

Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

Vertical "III-V" V-shaped nanomembranes epitaxially grown on a patterned Si[001] substrate and their enhanced light scattering.

We report on a new form of III-V compound semiconductor nanostructures growing epitaxially as vertical V-shaped nanomembranes on Si(001) and study their light-scattering properties. Precise position control of the InAs nanostructures in regular arrays is demonstrated by bottom-up synthesis using molecular beam epitaxy in nanoscale apertures on a SiO(2) mask. The InAs V-shaped nanomembranes are found to originate from the two opposite facets of a rectangular pyramidal island nucleus and extend along two opposite <111> B directions, forming flat {110} walls. Dark-field scattering experiments, in combination with light-scattering theory, show the presence of distinctive shape-dependent optical resonances significantly enhancing the local intensity of incident electromagnetic fields over tunable spectral regions. These new nanostructures could have interesting potential in nanosensors, infrared light emitters, and nonlinear optical elements.

Mechanism of homology recognition in DNA recombination from dual-molecule experiments.

In E. coli homologous recombination, a filament of RecA protein formed on DNA searches and pairs a homologous sequence within a second DNA molecule with remarkable speed and fidelity. Here, we directly probe the strength of the two-molecule interactions involved in homology search and recognition using dual-molecule manipulation, combining magnetic and optical tweezers. We find that the filament's secondary DNA-binding site interacts with a single strand of the incoming double-stranded DNA during homology sampling. Recognition requires opening of the helix and is strongly promoted by unwinding torsional stress. Recognition is achieved upon binding of both strands of the incoming dsDNA to each of two ssDNA-binding sites in the filament. The data indicate a physical picture for homology recognition in which the fidelity of the search process is governed by the distance between the DNA-binding sites.