Gasdermin D cysteine residues synergistically control its palmitoylation-mediated membrane targeting and assembly.

Gasdermin D (GSDMD) executes the cell death program of pyroptosis by assembling into oligomers that permeabilize the plasma membrane. Here, by single-molecule imaging, we elucidate the yet unclear mechanism of Gasdermin D pore assembly and the role of cysteine residues in GSDMD oligomerization. We show that GSDMD preassembles at the membrane into dimeric and trimeric building blocks that can either be inserted into the membrane, or further assemble into higher-order oligomers prior to insertion into the membrane. The GSDMD residues Cys39, Cys57, and Cys192 are the only relevant cysteines involved in GSDMD oligomerization. S-palmitoylation of Cys192, combined with the presence of negatively-charged lipids, controls GSDMD membrane targeting. Simultaneous Cys39/57/192-to-alanine (Ala) mutations, but not Ala mutations of Cys192 or the Cys39/57 pair individually, completely abolish GSDMD insertion into artificial membranes as well as into the plasma membrane. Finally, either Cys192 or the Cys39/Cys57 pair are sufficient to enable formation of GSDMD dimers/trimers, but they are all required for functional higher-order oligomer formation. Overall, our study unveils a cooperative role of Cys192 palmitoylation-mediated membrane binding and Cys39/57/192-mediated oligomerization in GSDMD pore assembly. This study supports a model in which Gasdermin D oligomerization relies on a two-step mechanism mediated by specific cysteine residues.

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters.

B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces, and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have used lattice light-sheet microscopy in combination with tailored custom-built 4D image analysis to study the cell-surface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM-BCR. Ramos B-cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membrane-localized IgM-BCR was found in clusters, which were mainly associated with the ridges and the microvilli. The dynamic ridge-network organization and the IgM-BCR cluster mobility were linked, and both were controlled by Arp2/3 complex activity. Our results suggest that dynamic topographical features of the cell surface govern the localization and transport of IgM-BCR clusters to facilitate antigen screening by B cells.

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria.

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.

Dendrite tapering actuates a self-organizing signaling circuit for stochastic filopodia initiation in neurons.

How signaling units spontaneously arise from a noisy cellular background is not well understood. Here, we show that stochastic membrane deformations can nucleate exploratory dendritic filopodia, dynamic actin-rich structures used by neurons to sample its surroundings for compatible transcellular contacts. A theoretical analysis demonstrates that corecruitment of positive and negative curvature-sensitive proteins to deformed membranes minimizes the free energy of the system, allowing the formation of long-lived curved membrane sections from stochastic membrane fluctuations. Quantitative experiments show that once recruited, curvature-sensitive proteins form a signaling circuit composed of interlinked positive and negative actin-regulatory feedback loops. As the positive but not the negative feedback loop can sense the dendrite diameter, this self-organizing circuit determines filopodia initiation frequency along tapering dendrites. Together, our findings identify a receptor-independent signaling circuit that employs random membrane deformations to simultaneously elicit and limit formation of exploratory filopodia to distal dendritic sites of developing neurons.

Computational resolution in single molecule localization - impact of noise level and emitter density.

Classical fluorescence microscopy is a powerful technique to image biological specimen under close-to-native conditions, but light diffraction limits its optical resolution to 200-300 nm-two orders of magnitude worse than the size of biomolecules. Assuming single fluorescent emitters, the final image of the optical system can be described by a convolution with the point spread function (PSF) smearing out details below the size of the PSF. In mathematical terms, fluorescence microscopy produces bandlimited space-continuous images that can be recovered from their spatial samples under the conditions of the classical Shannon-Nyquist theorem. During the past two decades, several single molecule localization techniques have been established and these allow for the determination of molecular positions with sub-pixel accuracy. Without noise, single emitter positions can be recovered precisely - no matter how close they are. We review recent work on the computational resolution limit with a sharp phase transition between two scenarios: 1) where emitters are well-separated with respect to the bandlimit and can be recovered up to the noise level and 2) closely distributed emitters which results in a strong noise amplification in the worst case. We close by discussing additional pitfalls using single molecule localization techniques based on structured illumination.

Reversible Live-Cell Labeling with Retro-engineered HaloTags Enables Long-Term High- and Super-Resolution Imaging.

Self-labeling enzymes (SLE) such as the HaloTag have emerged as powerful tools in high and super-resolution fluorescence microscopy. Newly developed fluorogenic SLE substrates enable imaging in the presence of excess dye. To exploit this feature for reversible labeling, we engineered two variants of HaloTag7 with restored dehalogenase activity. Kinetic studies in vitro showed different turnover kinetics for reHaloTagS (≈0.006 s-1 ) and reHaloTagF (≈0.055 s-1 ). Imaging by confocal and stimulated emission depletion microscopy yielded 3-5-time enhanced photostability of reHaloTag labeling. Prominently, single molecule imaging with reHaloTags enabled controlled and stable labeling density over extended time periods. By combination with structured illumination, simultaneous visualization of single molecule diffusion and organellar dynamics was achieved. These applications highlight the potential of reHaloTag labeling for pushing the limits of advanced fluorescence microscopy techniques.

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co-Condensation of Wnt Signalodroplets.

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of ≈300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.

Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex.

The mechanism and regulation of fusion between autophagosomes and lysosomes/vacuoles are still only partially understood in both yeast and mammals. In yeast, this fusion step requires SNARE proteins, the homotypic vacuole fusion and protein sorting (HOPS) tethering complex, the RAB7 GTPase Ypt7, and its guanine nucleotide exchange factor (GEF) Mon1-Ccz1. We and others recently identified Ykt6 as the autophagosomal SNARE protein. However, it has not been resolved when and how lipid-anchored Ykt6 is recruited onto autophagosomes. Here, we show that Ykt6 is recruited at an early stage of the formation of these carriers through a mechanism that depends on endoplasmic reticulum (ER)-resident Dsl1 complex and COPII-coated vesicles. Importantly, Ykt6 activity on autophagosomes is regulated by the Atg1 kinase complex, which inhibits Ykt6 through direct phosphorylation. Thus, our findings indicate that the Ykt6 pool on autophagosomal membranes is kept inactive by Atg1 phosphorylation, and once an autophagosome is ready to fuse with vacuole, Ykt6 dephosphorylation allows its engagement in the fusion event.

Engineered Upconversion Nanoparticles for Resolving Protein Interactions inside Living Cells.

Upconversion nanoparticles (UCNPs) convert near-infrared into visible light at much lower excitation densities than those used in classic two-photon absorption microscopy. Here, we engineered <50 nm UCNPs for application as efficient lanthanide resonance energy transfer (LRET) donors inside living cells. By optimizing the dopant concentrations and the core-shell structure for higher excitation densities, we observed enhanced UCNP emission as well as strongly increased sensitized acceptor fluorescence. For the application of these UCNPs in complex biological environments, we developed a biocompatible surface coating functionalized with a nanobody recognizing green fluorescent protein (GFP). Thus, rapid and specific targeting to GFP-tagged fusion proteins in the mitochondrial outer membrane and detection of protein interactions by LRET in living cells was achieved.

Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane.

Total internal reflection fluorescence (TIRF) microscopy offers powerful means to uncover the functional organization of proteins in the plasma membrane with very high spatial and temporal resolution. Traditional TIRF illumination, however, shows a Gaussian intensity profile, which is typically deteriorated by overlaying interference fringes hampering precise quantification of intensities-an important requisite for quantitative analyses in single-molecule localization microscopy (SMLM). Here, we combine flat-field illumination by using a standard πShaper with multi-angular TIR illumination by incorporating a spatial light modulator compatible with fast super-resolution structured illumination microscopy (SIM). This distinct combination enables quantitative multi-color SMLM with a highly homogenous illumination. By using a dual camera setup with optimized image splitting optics, we achieve a versatile combination of SMLM and SIM with up to three channels. We deploy this setup for establishing robust detection of receptor stoichiometries based on single-molecule intensity analysis and single-molecule Förster resonance energy transfer (smFRET). Homogeneous illumination furthermore enables long-term tracking and localization microscopy (TALM) of cell surface receptors identifying spatial heterogeneity of mobility and accessibility in the plasma membrane. By combination of TALM and SIM, spatially and molecularly heterogenous diffusion properties can be correlated with nanoscale cytoskeletal organization and dynamics.

Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold.

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.

PilT2 enhances the speed of gonococcal type IV pilus retraction and of twitching motility.

Type IV pilus (T4P) dynamics is important for various bacterial functions including host cell interaction, surface motility, and horizontal gene transfer. T4P retract rapidly by depolymerization, generating large mechanical force. The gene that encodes the pilus retraction ATPase PilT has multiple paralogues, whose number varies between different bacterial species, but their role in regulating physical parameters of T4P dynamics remains unclear. Here, we address this question in the human pathogen Neisseria gonorrhoeae, which possesses two pilT paralogues, namely pilT2 and pilU. We show that the speed of twitching motility is strongly reduced in a pilT2 deletion mutant, while directional persistence time and sensitivity of speed to oxygen are unaffected. Using laser tweezers, we found that the speed of single T4P retraction was reduced by a factor of ≈ 2 in a pilT2 deletion strain, whereas pilU deletion showed a minor effect. The maximum force and the probability for switching from retraction to elongation under application of high force were not significantly affected. We conclude that the physical parameters of T4P are fine-tuned through PilT2.

Lattice light-sheet microscopy and evaluation of dendritic transport in cultured hippocampal tissue reveal high variability in mobility of the KIF1A motor domain and entry into dendritic spines.

The unique morphology of neurons consists of a long axon and a highly variable arbour of dendritic processes, which assort neuronal cells into the main classes. The dendritic tree serves as the main domain for receiving synaptic input. Therefore, to maintain the structure and to be able to plastically change according to the incoming stimuli, molecules and organelles need to be readily available. This is achieved mainly via bi-directional transport of cargo along the microtubule lattices. Analysis of dendritic transport is lagging behind the investigation of axonal transport. Moreover, addressing transport mechanisms in tissue environment is very challenging and, therefore, rare. We employed high-speed volumetric lattice light-sheet microscopy and single particle tracking of truncated KIF1A motor protein lacking the cargo-binding domain. We focused our analysis on dendritic processes of CA1 pyramidal neurons in cultured hippocampal tissue. Analysis of individual trajectories revealed detailed information about stalling and high variability in movement and speed, and biased directionality of KIF1A. Furthermore, we could also observe KIF1A shortly entering into dendritic spines. We provide a workflow to analyse variations in the speed and direction of motor protein movement in dendrites that are either intrinsic properties of the motor domain or depend on the structure and modification of the microtubule trails.

Oxygen depletion triggers switching between discrete speed modes of gonococcal type IV pili.

Type IV pili are polymeric bacterial appendages that affect host cell interaction, motility, biofilm formation, and horizontal gene transfer. These force-generating motors work in at least three distinct velocity modes-elongation, and retraction at two distinct speeds, high and low. Yet it is unclear which regulatory inputs control their speeds. Here, we addressed this question for the human pathogen Neisseria gonorrhoeae. Using a combination of image analysis and surface analytics, we simultaneously monitored the speed of twitching motility and the concentration of oxygen. While oxygen was detectable, bacteria moved in the high-speed mode (1.5 µm/s). Upon full depletion of oxygen, gonococci simultaneously switched into the low-speed mode (0.5 µm/s). Speed switching was complete within seconds, independent of transcription, and reversible upon oxygen restoration. Using laser tweezers, we found that oxygen depletion triggered speed switching of the pilus motor at the single-molecule level. In the transition regime, single pili switched between both modes, indicating bistability. Switching is well described by a two-state model whereby the oxygen level controls the occupancy of the states.

Measuring forces between two single actin filaments during bundle formation.

Bundles of filamentous actin are dominant cytoskeletal structures, which play a crucial role in various cellular processes. As yet quantifying the fundamental interaction between two individual actin filaments forming the smallest possible bundle has not been realized. Applying holographic optical tweezers integrated with a microfluidic platform, we were able to measure the forces between two actin filaments during bundle formation. Quantitative analysis yields forces up to 0.2 pN depending on the concentration of bundling agents.

The HOPS tethering complex is required to maintain signaling endosome identity and TORC1 activity.

The endomembrane system of eukaryotic cells is essential for cellular homeostasis during growth and proliferation. Previous work showed that a central regulator of growth, namely the target of rapamycin complex 1 (TORC1), binds both membranes of vacuoles and signaling endosomes (SEs) that are distinct from multivesicular bodies (MVBs). Interestingly, the endosomal TORC1, which binds membranes in part via the EGO complex, critically defines vacuole integrity. Here, we demonstrate that SEs form at a branch point of the biosynthetic and endocytic pathways toward the vacuole and depend on MVB biogenesis. Importantly, function of the HOPS tethering complex is essential to maintain the identity of SEs and proper endosomal and vacuolar TORC1 activities. In HOPS mutants, the EGO complex redistributed to the Golgi, which resulted in a partial mislocalization of TORC1. Our study uncovers that SE function requires a functional HOPS complex and MVBs, suggesting a tight link between trafficking and signaling along the endolysosomal pathway.

Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane.

Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) receptor signaling complex in the plasma membrane of living cells by multicolor single-molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture.

A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy.

The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens.

Nanoscopic anatomy of dynamic multi-protein complexes at membranes resolved by graphene-induced energy transfer.

Insights into the conformational organization and dynamics of proteins complexes at membranes is essential for our mechanistic understanding of numerous key biological processes. Here, we introduce graphene-induced energy transfer (GIET) to probe axial orientation of arrested macromolecules at lipid monolayers. Based on a calibrated distance-dependent efficiency within a dynamic range of 25 nm, we analyzed the conformational organization of proteins and complexes involved in tethering and fusion at the lysosome-like yeast vacuole. We observed that the membrane-anchored Rab7-like GTPase Ypt7 shows conformational reorganization upon interactions with effector proteins. Ensemble and time-resolved single-molecule GIET experiments revealed that the HOPS tethering complex, when recruited via Ypt7 to membranes, is dynamically alternating between a 'closed' and an 'open' conformation, with the latter possibly interacting with incoming vesicles. Our work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub-nanometer resolution.

Proteins are part of the building blocks of life and are essential for structure, function and regulation of every cell, tissue and organ of the body. Proteins adopt different conformations to work efficiently within the various environments of a cell. They can also switch between shapes. One way to monitor how proteins change their shapes involves energy transfer. This approach can measure how close two proteins, or two parts of the same protein, are, by using dye labels that respond to each other when they are close together. For example, in a method called FRET, one dye label absorbs light and transfers the energy to the other label, which emits it as a different color of light. However, FRET only works over short distances (less than 10nm apart or 1/100,000th of a millimeter), so it is not useful for larger proteins. Here, Füllbrunn, Li et al. developed a method called GIET that uses graphene to analyze the dynamic structures of proteins on membrane surfaces. Graphene is a type of carbon nanomaterial that can absorb energy from dye labels and could provide a way to study protein interactions over longer distances. Graphene was deposited on a glass surface where it was coated with single layer of membrane, which could then be used to capture specific proteins. The results showed that GIET worked over longer distances (up to 30 nm) than FRET and could be used to study proteins attached to the membrane around graphene. Füllbrunn, Li et al. used it to examine a specific complex of proteins called HOPS, which is linked to multiple diseases, including Ebola, measuring distances between the head or tail of HOPS and the membrane to understand protein shapes. This revealed that HOPS adopts an upright position on membranes and alternates between open and closed shapes. The study of Füllbrunn, Li et al. highlights the ability of GIET to address unanswered questions about the function of protein complexes on membrane surfaces and sheds new light on the structural dynamics of HOPS in living cells. As it allows protein interactions to be studied over much greater distances, GIET could be a powerful new tool for cell biology research. Moreover, graphene is also useful in electron microscopy and both approaches combined could achieve a detailed structural picture of proteins in action.

Three-Dimensional Interfacing of Cells with Hierarchical Silicon Nano/Microstructures for Midinfrared Interrogation of In Situ Captured Proteins.

Label-free optical detection of biomolecules is currently limited by a lack of specificity rather than sensitivity. To exploit the much more characteristic refractive index dispersion in the mid-infrared (IR) regime, we have engineered three-dimensional IR-resonant silicon micropillar arrays (Si-MPAs) for protein sensing. By exploiting the unique hierarchical nano- and microstructured design of these Si-MPAs attained by CMOS-compatible silicon-based microfabrication processes, we achieved an optimized interrogation of surface protein binding. Based on spatially resolved surface functionalization, we demonstrate controlled three-dimensional interfacing of mammalian cells with Si-MPAs. Spatially controlled surface functionalization for site-specific protein immobilization enabled efficient targeting of soluble and membrane proteins into sensing hotspots directly from cells cultured on Si-MPAs. Protein binding to Si-MPA hotspots at submonolayer level was unambiguously detected by conventional Fourier transform IR spectroscopy. The compatibility with cost-effective CMOS-based microfabrication techniques readily allows integration of this novel IR transducer into fully fledged bioanalytical microdevices for selective and sensitive protein sensing.