RESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endocitose , Motivos de Aminoácidos , Animais , Transporte Biológico , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Hidrólise , Deleção de SequênciaRESUMO
The process of membrane fission is fundamental to diverse cellular processes such as nutrient uptake, synaptic transmission and organelle biogenesis, and it involves the localized application of curvature stress to a tubular membrane intermediate, forcing it to undergo scission. Alternative techniques for creating such substrates necessitate the use of micromanipulators or sophisticated optical traps and require a high level of technical expertise. We present a facile method to generate an array of membrane tubes supported on a passivated glass coverslip, which we refer to as supported membrane tubes (SMrTs). SMrT templates are formed upon hydration of a dry lipid mix in physiological buffer and subsequent flow-induced extrusion of the lipid reservoir into long membrane tubes with variable dimensions. Following surface passivation of coverslips, these templates can be formed from a variety of lipids, with as little as 1-2 nmol of lipid in a matter of 2 h, and can be used in membrane-curvature-sensitive fission assays.
Assuntos
Membrana Celular/metabolismo , Membranas Artificiais , Soluções Tampão , Vidro/química , Metabolismo dos Lipídeos , Fatores de TempoRESUMO
Classical dynamins bind the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate using the pleckstrin-homology domain (PHD) and engage in rapid membrane fission during synaptic vesicle recycling. This domain is conspicuously absent among extant bacterial and mitochondrial dynamins, however, where loop regions manage membrane recruitment. Inspired by the core design of bacterial and mitochondrial dynamins, we reengineered the classical dynamin by replacing its PHD with a polyhistidine or polylysine linker. Remarkably, when recruited via chelator or anionic lipids, respectively, the reengineered dynamin displayed the capacity to constrict and sever membrane tubes. However, when analyzed at single-event resolution, the tube-severing process displayed long-lived, highly constricted prefission intermediates that contributed to 10-fold reduction in bulk rates of membrane fission. Our results indicate that the PHD acts as a catalyst in dynamin-induced membrane fission and rationalize its adoption to meet the physiologic requirement of a fast-acting membrane fission apparatus.
Assuntos
Dinaminas/metabolismo , Dinaminas/ultraestrutura , Dinâmica Mitocondrial/fisiologia , Membrana Celular/metabolismo , Constrição , Dinaminas/genética , Endocitose/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Membranas/metabolismo , Membranas/fisiologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismo , Domínios de Homologia à Plecstrina/genética , Domínios de Homologia à Plecstrina/fisiologia , Domínios Proteicos , Estrutura Terciária de ProteínaRESUMO
Dynamin, the paradigmatic membrane fission catalyst, assembles as helical scaffolds that hydrolyse GTP to sever the tubular necks of clathrin-coated pits. Using a facile assay system of supported membrane tubes (SMrT) engineered to mimic the dimensions of necks of clathrin-coated pits, we monitor the dynamics of a dynamin-catalysed tube-severing reaction in real time using fluorescence microscopy. We find that GTP hydrolysis by an intact helical scaffold causes progressive constriction of the underlying membrane tube. On reaching a critical dimension of 7.3 nm in radius, the tube undergoes scission and concomitant splitting of the scaffold. In a constant GTP turnover scenario, scaffold assembly and GTP hydrolysis-induced tube constriction are kinetically inseparable events leading to tube-severing reactions occurring at timescales similar to the characteristic fission times seen in vivo. We anticipate SMrT templates to allow dynamic fluorescence-based detection of conformational changes occurring in self-assembling proteins that remodel membranes.
