A MAD7-based genome editing system for Escherichia coli.

A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genetic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA-guided nuclease MAD7 in Escherichia coli.

Clathrin coats partially preassemble and subsequently bend during endocytosis.

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites.

Maximum-likelihood model fitting for quantitative analysis of SMLM data.

Quantitative data analysis is important for any single-molecule localization microscopy (SMLM) workflow to extract biological insights from the coordinates of the single fluorophores. However, current approaches are restricted to simple geometries or require identical structures. Here, we present LocMoFit (Localization Model Fit), an open-source framework to fit an arbitrary model to localization coordinates.

The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial-DNA-mediated inflammation.

BAX and BAK are key apoptosis regulators that mediate the decisive step of mitochondrial outer membrane permeabilization. However, the mechanism by which they assemble the apoptotic pore remains obscure. Here, we report that BAX and BAK present distinct oligomerization properties, with BAK organizing into smaller structures with faster kinetics than BAX.

DRP1 interacts directly with BAX to induce its activation and apoptosis.

The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis.

How good are my data? Reference standards in superresolution microscopy.

Superresolution microscopy is becoming increasingly widespread in biological labs. While it holds enormous potential for biological discovery, it is a complex imaging technique that requires thorough optimization of various experimental parameters to yield data of the highest quality. Unfortunately, it remains challenging even for seasoned users to judge from the acquired images alone whether their superresolution microscopy pipeline is performing at its optimum, or if the image quality could be improved.

Publisher Correction: Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Topological data analysis quantifies biological nano-structure from single molecule localization microscopy.

Motivation: Localization microscopy data is represented by a set of spatial coordinates, each corresponding to a single detection, that form a point cloud. This can be analyzed either by rendering an image from these coordinates, or by analyzing the point cloud directly. Analysis of this type has focused on clustering detections into distinct groups which produces measurements such as cluster area, but has limited capacity to quantify complex molecular organization and nano-structure.

Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities.

Type-I myosins promote actin polymerization to drive membrane bending in endocytosis.

Clathrin-mediated endocytosis in budding yeast requires the formation of a dynamic actin network that produces the force to invaginate the plasma membrane against the intracellular turgor pressure. The type-I myosins Myo3 and Myo5 are important for endocytic membrane reshaping, but mechanistic details of their function remain scarce. Here, we studied the function of Myo3 and Myo5 during endocytosis using quantitative live-cell imaging and genetic perturbations.

Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization.

Three-dimensional single molecule localization microscopy relies on the fitting of the individual molecules with a point spread function (PSF) model. The reconstructed images often show local squeezing or expansion in . A common cause is depth-induced aberrations in conjunction with an imperfect PSF model calibrated from beads on a coverslip, resulting in a mismatch between measured PSF and real PSF.

Systematic Nanoscale Analysis of Endocytosis Links Efficient Vesicle Formation to Patterned Actin Nucleation.

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast.

Real-time 3D single-molecule localization using experimental point spread functions.

We present a real-time fitter for 3D single-molecule localization microscopy using experimental point spread functions (PSFs) that achieves minimal uncertainty in 3D on any microscope and is compatible with any PSF engineering approach. We used this method to image cellular structures and attained unprecedented image quality for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D super-resolution microscopy broadly accessible, even on standard microscopes without dedicated 3D optics.

Dual-Color and 3D Super-Resolution Microscopy of Multi-protein Assemblies.

Breaking the resolution limit of conventional microscopy by super-resolution microscopy (SRM) led to many new biological insights into protein assemblies at the nanoscale. Here we provide detailed protocols for single-molecule localization microscopy (SMLM) to image the structure of a protein complex. As examples, we show how to acquire single- and dual-color super-resolution images of the nuclear pore complex (NPC) and dual-color 3D data on actin and paxillin in focal adhesions.

Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores.

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy.

Visualizing the functional architecture of the endocytic machinery.

Clathrin-mediated endocytosis is an essential process that forms vesicles from the plasma membrane. Although most of the protein components of the endocytic protein machinery have been thoroughly characterized, their organization at the endocytic site is poorly understood. We developed a fluorescence microscopy method to track the average positions of yeast endocytic proteins in relation to each other with a time precision below 1 s and with a spatial precision of ~10 nm.

3D superresolution microscopy by supercritical angle detection.

We present a fundamentally new approach to 3D superresolution microscopy based on the principle of surface-generated fluorescence. This near-field fluorescence is strongly dependent on the distance of fluorophores from the coverslip and can therefore be used to estimate their axial positions. We established a robust and simple implementation of supercritical angle fluorescence detection for single-molecule localization microscopy, calibrated it using fluorescent bead samples, validated the method with DNA origami tetrahedra, and present proof-of-principle data on biological samples.

Localization microscopy in yeast.

Conventional light and fluorescence microscopy techniques have offered tremendous insight into cellular processes and structures. Their resolution is however intrinsically limited by diffraction. Superresolution techniques achieve an order of magnitude higher resolution.

LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes.

Seeing the big picture: Asymmetric macromolecular complexes that are NMR active in only a subset of their subunits can be prepared, thus decreasing NMR spectral complexity. For the hetero heptameric LSm1-7 and LSm2-8 rings NMR spectra of the individual subunits of the complete complex are obtained, showing a conserved RNA binding site. This LEGO-NMR technique makes large asymmetric complexes accessible to detailed NMR spectroscopic studies.

Structure of the LSm657 complex: an assembly intermediate of the LSm1-7 and LSm2-8 rings.

The nuclear LSm2-8 (like Sm) complex and the cytoplasmic LSm1-7 complex play a central role in mRNA splicing and degradation, respectively. The LSm proteins are related to the spliceosomal Sm proteins that form a heteroheptameric ring around small nuclear RNA. The assembly process of the heptameric Sm complex is well established and involves several smaller Sm assembly intermediates.

Measuring rotational diffusion of macromolecules by fluorescence correlation spectroscopy.

We describe a novel method to measure rotational diffusion of large biomolecules in solution based on fluorescence correlation on the nanosecond time scale. In contrast to conventional fluorescence anisotropy measurements, a correlation-based method will also work if the rotational diffusion time is much longer than the fluorescence decay time. Thus, the method is suited to study the rotational diffusion of macromolecules having rotational diffusion times of dozens to hundreds of nanoseconds, which is considerably larger than the fluorescence lifetime of most commercially available dyes or auto-fluorescent proteins.