Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy.

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

Self-quenched Fluorophore Dimers for DNA-PAINT and STED Microscopy.

Super-resolution techniques like single-molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non-covalent, weak affinity-based transient labelling systems. DNA-based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi-target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore-labelled single-stranded oligonucleotides. Here, we introduce short-distance self-quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2-fold. We characterise the optical and thermodynamic properties of fluorophore-dimer single-stranded DNA, and show super-resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background.

Multi-Color, Bleaching-Resistant Super-Resolution Optical Fluctuation Imaging with Oligonucleotide-Based Exchangeable Fluorophores.

Super-resolution optical fluctuation imaging (SOFI) is a super-resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background-free and show confocality and enhanced spatial resolution (super-resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore-labeled short DNA oligonucleotides, we labeled cellular structures with target-specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two-color imaging of cellular structures with SOFI.

Facilitating trypanosome imaging.

Research on trypanosomes as a model organism has provided a substantial contribution to a detailed understanding of basic cellular processes within the last few years. At the same time, major advances in super-resolution microscopy have been achieved, facilitating the resolution of biological structures in living cells at a scale of a few nm. However, the motility of trypanosomes has prevented access to high resolution microscopy of live cells. Here, we present a hydrogel based on poly(ethylene glycol) functionalized with either norbornene or thiol moieties for UV induced thiol-ene crosslinking for the embedding and imaging of live trypanosomes. The resulting gel exhibits low autofluorescence properties, immobilizes the cells efficiently on the nanometer scale and is compatible with cell viability for up to one hour at 24 °C. We applied super-resolution imaging to the inner plasma membrane leaflet using lipid-anchored eYFP as a probe. We find specific domains within the membrane where the fluorescence either accumulates or appears diluted rather than being homogenously distributed. Based on a Ripley's analysis, the size of the domains was determined to be raccumulated=170±5 nm and rdilute>115±15 nm. We hypothesize that this structuring of the membrane is associated with the underlying cytoskeleton.

Self-Quenched Fluorophore-DNA Labels for Super-Resolution Fluorescence Microscopy.

Protein labeling through transient and repetitive hybridization of short, fluorophore-labeled DNA oligonucleotides has become widely applied in various optical super-resolution microscopy methods. The main advantages are multitarget imaging and molecular quantification. A challenge is the high background signal originating from the presence of unbound fluorophore-DNA labels in solution. Here, we report the self-quenching of fluorophore dimers conjugated to DNA oligonucleotides as a general concept to reduce the fluorescence background. Upon hybridization, the fluorescence signals of both fluorophores are restored. We expand the toolbox of fluorophores suitable for self-quenching and report their spectra and hybridization equilibria. We apply self-quenched fluorophore-DNA labels to stimulated emission depletion microscopy and single-molecule localization microscopy and report improved imaging performances.

The function of ER-phagy receptors is regulated through phosphorylation-dependent ubiquitination pathways.

Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and essential to maintain cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a critical role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyze phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy upon mTOR (mechanistic target of rapamycin) inhibition. An unbiased screen of kinase inhibitors reveals CK2 to be essential for FAM134B- and FAM134C-driven ER-phagy after mTOR inhibition. Furthermore, we provide evidence that ER-phagy receptors are regulated by ubiquitination events and that treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Using super-resolution microscopy, we show that CK2 activity is essential for the formation of high-density FAM134B and FAM134C clusters. In addition, dense clustering of FAM134B and FAM134C requires phosphorylation-dependent ubiquitination of FAM134B and FAM134C. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of FAM134B and FAM134C phosphosites prevents ubiquitination of FAM134 proteins, formation of high-density clusters, as well as Torin1-induced ER-phagy flux. Therefore, we propose that CK2-dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.

Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy.

Investigating the interplay of cellular proteins with optical microscopy requires multitarget labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging experiment. Advanced microscopy methods such as STED microscopy additionally demand balanced excitation, depletion, and emission wavelengths for all fluorophores, further reducing multiplexing capabilities. Noncovalent, weak-affinity labels bypass this "spectral barrier" through label exchange and sequential imaging of different targets. Here, we combine exchangeable HaloTag ligands, weak-affinity DNA hybridization, and hydrophophic and protein-peptide interactions to increase labeling flexibility and demonstrate six-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching as well as facilitate long acquisition times and multicolor live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increases the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images.

Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection.

SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.

Structural basis for the shielding function of the dynamic trypanosome variant surface glycoprotein coat.

The most prominent defence of the unicellular parasite Trypanosoma brucei against the host immune system is a dense coat that comprises a variant surface glycoprotein (VSG). Despite the importance of the VSG family, no complete structure of a VSG has been reported. Making use of high-resolution structures of individual VSG domains, we employed small-angle X-ray scattering to elucidate the first two complete VSG structures. The resulting models imply that the linker regions confer great flexibility between domains, which suggests that VSGs can adopt two main conformations to respond to obstacles and changes of protein density, while maintaining a protective barrier at all times. Single-molecule diffusion measurements of VSG in supported lipid bilayers substantiate this possibility, as two freely diffusing populations could be detected. This translates into a highly flexible overall topology of the surface VSG coat, which displays both lateral movement in the plane of the membrane and variation in the overall thickness of the coat.

N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold.

The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed.

The molecular size of the extra-membrane domain influences the diffusion of the GPI-anchored VSG on the trypanosome plasma membrane.

A plethora of proteins undergo random and passive diffusion in biological membranes. While the contribution of the membrane-embedded domain to diffusion is well established, the potential impact of the extra-membrane protein part has been largely neglected. Here, we show that the molecular length influences the diffusion coefficient of GPI-anchored proteins: smaller proteins diffuse faster than larger ones. The distinct diffusion properties of differently sized membrane proteins are biologically relevant. The variant surface glycoprotein (VSG) of African trypanosomes, for example, is sized for an effective diffusion-driven randomization on the cell surface, a process that is essential for parasite virulence. We propose that the molecular sizes of proteins dominating the cell surfaces of other eukaryotic pathogens may also be related to diffusion-limited functions.