Autophagy recycles cytoplasmic components by sequestering them in double membrane–surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.

After uptake into target cells, many nonenveloped viruses undergo conformational changes in the low-pH environment of the endocytic compartment. This results in exposure of amphipathic viral peptides and/or hydrophobic protein domains that are inserted into and either disrupt or perforate the vesicular membranes. The viral nucleic acids thereby gain access to the cytosol and initiate replication. We here demonstrate the in vitro transfer of the single-stranded positive-sense RNA genome of human rhinovirus 2 into liposomes decorated with recombinant very-low-density lipoprotein receptor fragments. Membrane-attached virions were exposed to pH 5.4, mimicking the in vivo pH environment of late endosomes. This triggered the release of the RNA whose arrival in the liposomal lumen was detected via in situ cDNA synthesis by encapsulated reverse transcriptase. Subsequently, cDNA was PCR amplified. At a low ratio between virions and lipids, RNA transfer was positively correlated with virus concentration. However, membranes became leaky at higher virus concentrations, which resulted in decreased cDNA synthesis. In accordance with earlier in vivo data, the RNA passes through the lipid membrane without causing gross damage to vesicles at physiologically relevant virus concentrations.

Therapeutic and diagnostic nucleic acid aptamers are designed to bind tightly and specifically to their target. The combination of structural and kinetic analyses of aptamer interactions has gained increasing importance. Here, we present a fluorescence-based switchSENSE aptasensor for the detailed kinetic characterization of aptamer–analyte interaction and aptamer folding, employing the thrombin-binding aptamer (TBA) as a model system. Thrombin-binding aptamer folding into a G-quadruplex and its binding to thrombin strongly depend on the type and concentration of ions present in solution. We observed conformational changes induced by cations in real-time and determined the folding and unfolding kinetics of the aptamer. The aptamer’s affinity for K+ was found to be more than one order of magnitude higher than for other cations (K+ > NH4+ >> Na+ > Li+). The aptamer’s affinity to its protein target thrombin in the presence of different cations followed the same trend but differed by more than three orders of magnitude (KD = 0.15 nM to 250 nM). While the stability (kOFF) of the thrombin–TBA complex was similar in all conditions, the cation type strongly influenced the association rate (kON). These results demonstrated that protein–aptamer binding is intrinsically related to the correct aptamer fold and, hence, to the presence of stabilizing ions. Because fast binding kinetics with on-rates exceeding 108 M−1s−1 can be quantified, and folding-related phenomena can be directly resolved, switchSENSE is a useful analytical tool for in-depth characterization of aptamer–ion and aptamer–protein interactions.

The binding properties of bispecific antibodies (bsAb) are crucial for their function, especially when two antigens are targeted on the same cell surface. Dynamic interactions between each of the antibody’s arms and its cognate target cause the formation and decay of a biologically functional ternary complex. How association and dissociation processes work cooperatively, and how they influence the avidity of the ternary complex, is still poorly understood. Here, we present a biosensor assay for the simultaneous measurement of the binding kinetics of the therapeutic bsAb emicizumab (Hemlibra®) and its two targets, the blood coagulation factors IX and X (FIX, FX). We describe an automated workflow to characterize binary and ternary-binding modes, utilizing a Y-shaped DNA nanostructure to immobilize the antigens on a sensor and to emulate conditions on a cell or platelet surface by presenting the antigens with optimal accessibility for the bsAb flown over the sensor as analyte. We find that emicizumab binds FX much stronger than FIX (K d = 0.05 µM vs. 5 µM, t 1/2 = 20 s vs. 1 s) with profound consequences on the avidity of the ternary complex, which is dominated by FX’s binding properties and a hand-off mechanism from FX to FIX. Moreover, formation and decay of the ternary complex depend on the bsAb concentration during the association phase. Emicizumab’s in-vivo mode of action and the catalytic activation of FX can be rationalized from the analyzed binding kinetics. The assay and workflow are well suited for the screening of bispecific binders in drug discovery and provide valuable new kinetic information. Abbreviations: bsAb: bispecific antibody; FVIII/FIX/FX: coagulation factors VIII/IX/X; SPR: surface plasmon resonance; k on : association rate constant; k off : dissociation rate constant; K D : equilibrium dissociation constant; t 1/2 : dissociation half-life

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation—a starting point for further studies with cell and animal models of SCA3.