A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha- synuclein to lysosomes

The nervous system spread of alpha-synuclein fibrils leads to Parkinson′s disease (PD) and other synucleinopathies, yet the mechanisms underlying internalization and cell-to-cell transfer are enigmatic. Here we use confocal and superresolution microscopy, subcellular fractionation and electron microscopy of immunogold labelled alpha-synuclein pre-formed fibrils (PFF) to demonstrate that this toxic protein species enters cells using a novel form of ultra-rapid macropinocytosis with transfer to lysosomes in as little as 2 minutes, an unprecedented cell biological kinetic for lysosomal targeting. PFF uptake circumvents classical endosomal pathways and is independent of clathrin. Immunogold-labelled PFF are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, and in lysosomes. While many of the fibrils remain in lysosomes that continue to take up PFF for hours, a portion are transferred to neighboring naive cells on the external face of vesicles, likely exosomes. These data indicate that PFF uses a novel internalization mechanism as a component of cell-to-cell propagation.

Precise measurement of nanoscopic septin ring structures in deep learning-assisted quantitative superresolution microscopy

The combination of image analysis and fluorescence superresolution microscopy methods allows for unprecedented insight into the organization of macromolecular assemblies in cells. Advances in deep learning-based object recognition enables the automated processing of large amounts of data, resulting in high accuracy through averaging. However, while the analysis of highly symmetric structures of constant size allows for a resolution approaching the dimensions of structural biology, deep learning methods are prone to different forms of bias. A biased recognition of structures may prohibit the development of readouts for processes that involve significant changes in size or shape of amorphous macromolecular complexes. What is required to overcome this problem is a detailed investigation of potential sources of bias and the rigorous testing of trained models using real or simulated data covering a wide dynamic range of possible results. Here we combine single molecule localization-based superresolution microscopy of septin ring structures with the training of several different deep learning models for a quantitative investigation of bias resulting from different training approaches and finally quantitative changes in septin ring structures. We find that trade-off exists between measurement accuracy and the dynamic range of recognized phenotypes. Using our trained models, we furthermore find that septin ring size can be explained by the number of subunits they are assembled from alone. Our work provides a new experimental system for the investigation of septin polymerization.

Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions

Epithelial cells assemble specialized actomyosin structures at E-Cadherin–based cell–cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.

A general design of caging-group free photoactivatable fluorophores for live-cell nanoscopy

The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every superresolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging superresolution methods. Photoactivatable dyes offer significant improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogs, yielding a new family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell fluorescence microscopy, and both coordinate-targeted stimulated emission depletion (STED) and coordinate-stochastic single-molecule localization superresolution microscopy (SMLM).

Particle localization using local gradients and its application to nanometer stabilization of a microscope

Accurate localization of single particles plays an increasingly important role in a range of biological techniques, including single molecule tracking and localization-based superresolution microscopy. Such techniques require fast and accurate particle localization algorithms as well as nanometer-scale stability of the microscope. Here, we present a universal method for three-dimensional localization of single labeled and unlabeled particles based on local gradient calculation of microscopy images. The method outperforms current techniques in high noise conditions, and it is capable of nanometer accuracy localization of nano- and micro-particles with sub-ms calculation time. By localizing a fixed particle as fiducial mark and running a feedback loop, we demonstrate its applicability for active drift correction in sensitive nanomechanical measurements such as optical trapping and superresolution imaging. A multiplatform open software package comprising a set of tools for local gradient calculation in brightfield and fluorescence microscopy is shared to the scientific community.

Substrate rigidity modulates traction forces and stoichiometry of cell matrix adhesions.

In cell matrix adhesions, integrin receptors and associated proteins provide a dynamic coupling of the extracellular matrix (ECM) to the cytoskeleton. This allows bidirectional transmission of forces between the ECM and the cytoskeleton, which tunes intracellular signaling cascades that control survival, proliferation, differentiation, and motility. The quantitative relationships between recruitment of distinct cell matrix adhesion proteins and local cellular traction forces are not known. Here, we applied quantitative superresolution microscopy to cell matrix adhesions formed on fibronectin-stamped elastomeric pillars and developed an approach to relate the number of talin, vinculin, paxillin, and focal adhesion kinase (FAK) molecules to the local cellular traction force. We find that FAK recruitment does not show an association with traction-force application whereas a ~60 pN force increase is associated with the recruitment of one talin, two vinculin, and two paxillin molecules on a substrate of effective stiffness of 47 kPa. On a substrate with a four-fold lower effective stiffness the stoichiometry of talin:vinculin:paxillin changes to 2:12:6 for the same ~60 pN traction force. The relative change in force-related vinculin recruitment indicates a stiffness-dependent switch in vinculin function in cell matrix adhesions. Our results reveal a substrate-stiffness-dependent modulation of the relation between cellular traction-force and the molecular stoichiometry of cell-matrix adhesions.

Superresolution microscopy reveals partial preassembly and subsequent bending of the clathrin coat during endocytosis

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargos. 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 endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis and find that clathrin coats assemble first on flat membranes to ~50% of the coat area, before they become rapidly and continuously bent. We introduce a mathematical model that assumes a positive feedback for curvature generation of the clathrin coat. This Cooperative Curvature Model agrees excellently with experimental data in three cell lines, and likely describes a general pathway for clathrin coat remodeling during endocytosis.

The Dictyostelium Centrosome

The centrosome of Dictyostelium amoebae contains no centrioles and consists of a cylindrical layered core structure surrounded by a corona harboring microtubule-nucleating γ-tubulin complexes. It is the major centrosomal model beyond animals and yeasts. Proteomics, protein interaction studies by BioID and superresolution microscopy methods led to considerable progress in our understanding of the composition, structure and function of this centrosome type. We discuss all currently known components of the Dictyostelium centrosome in comparison to other centrosomes of animals and yeasts.

The human cytomegalovirus protein pUL13 targets mitochondrial cristae architecture to increase cellular respiration during infection

Viruses modulate mitochondrial processes during infection to increase biosynthetic precursors and energy output, fueling virus replication. In a surprising fashion, although it triggers mitochondrial fragmentation, the prevalent pathogen human cytomegalovirus (HCMV) increases mitochondrial metabolism through a yet-unknown mechanism. Here, we integrate molecular virology, metabolic assays, quantitative proteomics, and superresolution confocal microscopy to define this mechanism. We establish that the previously uncharacterized viral protein pUL13 is required for productive HCMV replication, targets the mitochondria, and functions to increase oxidative phosphorylation during infection. We demonstrate that pUL13 forms temporally tuned interactions with the mitochondrial contact site and cristae organizing system (MICOS) complex, a critical regulator of cristae architecture and electron transport chain (ETC) function. Stimulated emission depletion superresolution microscopy shows that expression of pUL13 alters cristae architecture. Indeed, using live-cell Seahorse assays, we establish that pUL13 alone is sufficient to increase cellular respiration, not requiring the presence of other viral proteins. Our findings address the outstanding question of how HCMV targets mitochondria to increase bioenergetic output and expands the knowledge of the intricate connection between mitochondrial architecture and ETC function.