Mesoscale physics bridges the gap between the microscopic degrees of freedom of a system and its large-scale continuous behavior and highlights the role of a few key quantities in complex and multiscale phenomena, like dynamin-driven fission of lipid membranes. The dynamin protein wraps the neck formed during clathrin-mediated endocytosis, for instance, and constricts it until severing occurs. Although ubiquitous and fundamental for life, the cooperation between the GTP-consuming conformational changes within the protein and the full-scale response of the underlying lipid substrate is yet to be unraveled. In this work, we build an effective mesoscopic model from constriction to fission of lipid tubules based on continuum membrane elasticity and implicitly accounting for ratchet-like power strokes of dynamins. Localization of the fission event, the overall geometry, and the energy expenditure we predict comply with the major experimental findings. This bolsters the idea that a continuous picture emerges soon enough to relate dynamin polymerization length and membrane rigidity and tension with the optimal pathway to fission. We therefore suggest that dynamins found in in vivo processes may optimize their structure accordingly. Ultimately, we shed light on real-time conductance measurements available in literature and predict the fission time dependency on elastic parameters.

Membrane tubulation coupled with fission (MTCF) is a widespread phenomenon but mechanisms for their coordination remain unclear, partly because of the lack of assays to monitor dynamics of membrane tubulation and subsequent fission. Using polymer cushioned bilayer islands, we analyze the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal this mixture to constitute a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 facilitates recruitment but inhibits membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission.

The functions of human Apolipoproteins L (APOLs) are poorly understood, but involve diverse activities like lysis of bloodstream trypanosomes and intracellular bacteria, modulation of viral infection and induction of apoptosis, autophagy, and chronic kidney disease. Based on recent work, I propose that the basic function of APOLs is the control of membrane dynamics, at least in the Golgi and mitochondrion. Together with neuronal calcium sensor-1 (NCS1) and calneuron-1 (CALN1), APOL3 controls the activity of phosphatidylinositol-4-kinase-IIIB (PI4KB), involved in both Golgi and mitochondrion membrane fission. Whereas secreted APOL1 induces African trypanosome lysis through membrane permeabilization of the parasite mitochondrion, intracellular APOL1 conditions non-muscular myosin-2A (NM2A)-mediated transfer of PI4KB and APOL3 from the Golgi to the mitochondrion under conditions interfering with PI4KB-APOL3 interaction, such as APOL1 C-terminal variant expression or virus-induced inflammatory signalling. APOL3 controls mitophagy through complementary interactions with the membrane fission factor PI4KB and the membrane fusion factor vesicle-associated membrane protein-8 (VAMP8). In mice, the basic APOL1 and APOL3 activities could be exerted by mAPOL9 and mAPOL8, respectively. Perspectives regarding the mechanism and treatment of APOL1-related kidney disease are discussed, as well as speculations on additional APOLs functions, such as APOL6 involvement in adipocyte membrane dynamics through interaction with myosin-10 (MYH10).

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.

Artificial or synthetic organelles are a key challenge for bottom-up synthetic biology. So far, synthetic organelles have typically been based on spherical membrane compartments, used to spatially confine selected chemical reactions. In vivo, these compartments are often far from being spherical and can exhibit rather complex architectures. A particularly fascinating example is provided by the endoplasmic reticulum (ER), which extends throughout the whole cell by forming a continuous network of membrane nanotubes connected by three-way junctions. The nanotubes have a typical diameter of between 50 and 100 nm. In spite of much experimental progress, several fundamental aspects of the ER morphology remain elusive. A long-standing puzzle is the straight appearance of the tubules in the light microscope, which form irregular polygons with contact angles close to 120°. Another puzzling aspect is the nanoscopic shapes of the tubules and junctions, for which very different images have been obtained by electron microcopy and structured illumination microscopy. Furthermore, both the formation and maintenance of the reticular networks require GTP and GTP-hydrolyzing membrane proteins. In fact, the networks are destroyed by the fragmentation of nanotubes when the supply of GTP is interrupted. Here, it is argued that all of these puzzling observations are intimately related to each other and to the dimerization of two membrane proteins anchored to the same membrane. So far, the functional significance of this dimerization process remained elusive and, thus, seemed to waste a lot of GTP. However, this process can generate an effective membrane tension that stabilizes the irregular polygonal geometry of the reticular networks and prevents the fragmentation of their tubules, thereby maintaining the integrity of the ER. By incorporating the GTP-hydrolyzing membrane proteins into giant unilamellar vesicles, the effective membrane tension will become accessible to systematic experimental studies.

Atg18, Atg21 and Hsv2 are homologous β-propeller proteins binding to PI3P and PI(3,5)P2. Atg18 is thought to organize lipid transferring protein complexes at contact sites of the growing autophagosome (phagophore) with both the ER and the vacuole. Atg21 is restricted to the vacuole phagophore contact, where it organizes part of the Atg8-lipidation machinery. The role of Hsv2 is less understood, it partly affects micronucleophagy. Atg18 is further involved in regulation of PI(3,5)P2 synthesis. Recently, a novel Atg18-retromer complex and its role in vacuole homeostasis and membrane fission was uncovered.

Mitochondria are dynamic organelles that undergo cycles of fission and fusion at a unified platform defined by endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCSs). These MCSs or nodes co-localize fission and fusion machinery. We set out to identify how ER-associated mitochondrial nodes can regulate both fission and fusion machinery assembly. We have used a promiscuous biotin ligase linked to the fusion machinery, Mfn1, and proteomics to identify an ER membrane protein, ABHD16A, as a major regulator of node formation. In the absence of ABHD16A, fission and fusion machineries fail to recruit to ER-associated mitochondrial nodes, and fission and fusion rates are significantly reduced. ABHD16A contains an acyltransferase motif and an α/β hydrolase domain, and point mutations in critical residues of these regions fail to rescue the formation of ER-associated mitochondrial hot spots. These data suggest a mechanism whereby ABHD16A functions by altering phospholipid composition at ER-mitochondria MCSs. Our data present the first example of an ER membrane protein that regulates the recruitment of both fission and fusion machineries to mitochondria.

Membrane-enclosed transport carriers sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of eukaryotic organisms. While much is known about the formation and release of carriers from specific intracellular membranes, the mechanism of carrier formation from the recycling endosome, a compartment central to cellular signaling, remains to be resolved. In Caenorhabditis elegans, formation of transport carriers from the recycling endosome requires the dynamin-like, Eps15-homology domain (EHD) protein, RME-1, functioning with the Bin/Amphiphysin/Rvs (N-BAR) domain protein, AMPH-1. Here we show, using a free-solution single-particle technique known as burst analysis spectroscopy (BAS), that AMPH-1 alone creates small, tubular-vesicular products from large, unilamellar vesicles by membrane fission. Membrane fission requires the amphipathic H0 helix of AMPH-1 and is slowed in the presence of RME-1. Unexpectedly, AMPH-1-induced membrane fission is stimulated in the presence of GTP. Furthermore, the GTP-stimulated membrane fission activity seen for AMPH-1 is recapitulated by the heterodimeric N-BAR amphiphysin protein from yeast, Rvs161/167p, strongly suggesting that GTP-stimulated membrane fission is a general property of this important class of N-BAR proteins.

Intercellular communication is an essential process in all multicellular organisms. During this process, molecules secreted by one cell will bind to a receptor on the cognate cell leading to the subsequent uptake of the receptor-ligand complex. Once inside, the cell then determines the fate of the receptor-ligand complex and any other proteins that were endocytosed together. Approximately 80% of endocytosed material is recycled back to the plasma membrane either directly or indirectly via the Golgi apparatus and the remaining 20% is delivered to the lysosome for degradation. Although most pathways have been identified, we still lack understanding on how specificity in sorting of recycling cargos into different pathways is achieved, and how the cell reaches high accuracy of these processes in the absence of clear sorting signals in the bulk of the client proteins. In this review, we will summarize our current understanding of the mechanism behind recycling cargo sorting and propose a model of differential affinities between cargo and cargo receptors/adaptors with regards to iterative sorting in endosomes.