Markus Mund

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. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc‐shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.

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. With these data, integrated with shapes of endocytic membrane intermediates and with superresolution imaging, we could visualize the dynamic architecture of the endocytic machinery. We showed how different coat proteins are distributed within the coat structure and how the assembly dynamics of N-BAR proteins relate to membrane shape changes. Moreover, we found that the region of actin polymerization is located at the base of the endocytic invagination, with the growing ends of filaments pointing toward the plasma membrane. DOI: http://dx.doi.org/10.7554/eLife.04535.001

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. Among these, localization microscopy relies on the position determination of single emitters with nanometer accuracy, which allows the subsequent reconstruction of an image of the target structure. In this chapter, we provide general guidelines for localization microscopy with a focus on Saccharomyces cerevisiae. Its different cellular architecture complicates efforts to directly transfer protocols established in mammalian cells to yeast. We compare different methodologies to label structures of interest and provide protocols for the respective sample preparation, which are not limited to yeast. Using these guidelines, nanoscopic subcellular structures in yeast can be investigated by localization microscopy, which perfectly complements live-cell fluorescence and electron microscopy.

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.

Systematic analysis of the molecular architecture of endocytosis reveals a nanoscale actin nucleation template that drives efficient vesicle formation

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. We found that proteins assemble by radially-ordered recruitment according to function. WASP family proteins form a circular nano-scale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template creates sufficient force for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

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

The study of complex molecular organisation and nanostructure by localization based microscopy is limited by the available analysis tools. We present a segmentation protocol which, through the application of persistence based clustering, is capable of probing densely packed structures which vary in scale. An increase in segmentation performance over state-of-the-art methods is demonstrated. Moreover we employ persistence homology to move beyond clustering, and quantify the topological structure within data. This provides new information about the preserved shapes formed by molecular architecture. Our methods are flexible and we demonstrate this by applying them to receptor clustering in platelets, nuclear pore components and endocytic proteins. Both 2D and 3D implementations are provided within RSMLM, an R package for pointillist based analysis and batch processing of localization microscopy data.

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.

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

Summary 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. We found that proteins assemble by radially ordered recruitment according to function. WASP family proteins form a circular nanoscale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

Fast, robust and precise 3D localization for arbitrary point spread functions

We present a fitter for 3D single-molecule localization of arbitrary, experimental point spread functions (PSFs) that reaches minimum uncertainty for EMCCD and sCMOS cameras, and achieves more than 105 fits/s. We provide tools to robustly model experimental PSFs and correct for depth induced aberrations, which allowed us to achieve an unprecedented 3D resolution with engineered astigmatic PSFs, and acquire high quality 3D superresolution images even on standard microscopes without 3D optics.

Superresolution Imaging of Endocytic Structures in S. Cerevisiae

Endocytosis is a highly intricate cellular process, which in yeast involves the ordered recruitment and disassembly of around 60 proteins. Live-cell microscopy has lead to tremendous insight into composition and dynamics of the endocytic machinery. Due to the diffraction limit however, the maximum resolution of around 250 nm was much above the relevant endocytic size range. Electron microscopy on the other hand offers nanometer resolution, but lacks molecular specificity. As a result, structural features of most endocytic protein assemblies are largely unknown.We employ localization microscopy (PALM/STORM) to study endocytic structures in S. cerevisiae. This method is ideal to study static structures, which is why cells are typically fixated during sample preparation, leading to the loss of temporal resolution. We address this issue by recording snapshots of all key intermediate steps during endocytosis. For each fixated endocytic site, we can pinpoint the progress along the endocytic timeline by correlating information from marker proteins with a distinct temporal recruitment pattern.Our current efforts focus on the intermediate and late coat assembly preceding scission. Here, we were able to reveal subdiffraction features regarding shape and structure of endocytic coat proteins that were previously inaccessible. By visualizing many proteins pairs with dual-color superresolution microscopy, we are pursuing to obtain a comprehensive structural picture of the endocytic proteome.