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Endocytic and recycling pathways generate cargo-laden transport carriers by membrane fission. Classical dynamins, which generate transport carriers during endocytosis, constrict and cause fission of membrane tubes in response to GTP hydrolysis. Relatively, less is known about the ATP-binding Eps15-homology domain-containing protein1 (EHD1), a dynamin family member that functions at the endocytic-recycling compartment. Here, we show using cross complementation assays in C. elegans that EHD1’s membrane binding and ATP hydrolysis activities are necessary for endocytic recycling. Further, we show that ATP-bound EHD1 forms membrane-active scaffolds that bulge tubular model membranes. ATP hydrolysis promotes scaffold self-assembly, causing the bulge to extend and thin down intermediate regions on the tube. On tubes below 25 nm in radius, such thinning leads to scission. Molecular dynamics simulations corroborate this scission pathway. Deletion of N-terminal residues causes defects in stable scaffolding, scission and endocytic recycling. Thus, ATP hydrolysis-dependent membrane remodeling links EHD1 functions to endocytic recycling.

The quantification of membrane-associated biomolecular interactions is crucial to our understanding of various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition of fluorescent labels, which complicates the quantification of the involved stoichiometries and dynamics because of low temporal resolution and the inherent limitations associated with labeling efficiency, photoblinking and photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method to the membrane remodeling GTPase, dynamin-1, reveals heterogeneous mixtures of dimer-based oligomers, oligomer-dependent mobilities, membrane affinities and (dis)association of individual complexes. These capabilities, together with assay-based advances for studying integral membrane proteins, will enable the elucidation of biomolecular mechanisms in and on lipid bilayers.

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RBR domains, a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.

Summary Protein oligomerization is central to biological function and regulation, yet its experimental quantification and measurement of dynamic transitions in solution remain challenging. Here, we show that single molecule mass photometry quantifies affinity and polydispersity of heterogeneous protein complexes in solution. We demonstrate these capabilities by studying the functionally relevant oligomeric equilibria of 2-cysteine peroxiredoxins (2CPs). Comparison of the polydispersity of plant and human 2CPs as a function of concentration and redox state revealed features conserved among all 2CPs. In addition, we also find species-specific differences in oligomeric transitions, the occurrence of intermediates and the formation of high molecular weight complexes, which are associated with chaperone activity or act as a storage pool for more efficient dimers outlining the functional differentiation of human 2CPs. Our results point to a diversified functionality of oligomerization for 2CPs and illustrate the power of mass photometry for characterizing heterogeneous oligomeric protein distributions in near native conditions.

We introduce dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of membrane-associated proteins. Our method enables quantitative studies of their mobility, membrane affinity and interactions at the single molecule level. Application to the membrane remodelling GTPase dynamin1 reveals heterogeneous mixtures of oligomers suggesting that the fundamental building block for oligomerisation is a dimer, challenging current tetramer-centric models. Dynamic mass photometry has the ability to transform our approach to studying biomolecular mechanisms in and on lipid bilayers.

Single molecule mass photometry was used to study the dynamic equilibria of the ubiquitous and highly abundant 2-Cysteine peroxiredoxins (2-CysPRX). 2-CysPRXs adopt distinct functions in all cells dependent on their oligomeric conformation ranging from dimers to decamers and high molecular weight aggregates (HMW). The oligomeric state depends on the redox state of their catalytic cysteinyl residues. To which degree they interconvert, how the interconversion is regulated, and how the oligomerisation propensity is organism specific remains, however, poorly understood. The dynamics differs between wild-type and single point mutants affecting the oligomerization interfaces, with concomitant changes to function. Titrating concentration and redox state of Arabidopsis thaliana and human 2-CysPRXs revealed features conserved among all 2-CysPRX and clear differences concerning oligomer transitions, the occurrence of transition states and the formation of HMW which are associated with chaperone activity or storage. The results indicate functional differentiation of human 2-CysPRXs. Our results point to a diversified functionality of oligomerization for 2-CysPRXs and illustrate the power of mass photometry to non-invasively quantify oligomer distributions in a redox environment. This knowledge is important to fully address and model PRX function in cell redox signaling e.g., in photosynthesis, cardiovascular and neurological diseases or carcinogenesis.

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