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Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate (1,2), characterized by a 'stalk' in which only the outer membrane monolayers of the two compartments have merged to form a localized non-bilayer connection (1-3). Formation of the hemi-fission intermediate requires energy input from proteins catalysing membrane remodelling; however, the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analysed how the GTPase cycle of human dynamin 1, the prototypical membrane fission catalyst (4-6), is directly coupled to membrane remodelling. We used intramolecular chemical crosslinking to stabilize dynamin in its GDP x Al[F.sub.4.sup.-] -bound transition state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemifission. A second mode of dynamin activity, fuelled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent, drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction (7,8), the force bimodality might constitute a general paradigm for leakage-free membrane remodelling., Membrane fission and fusion both involve a pivotal stage, in which lipids rapidly rearrange into a new topology under extreme protein-driven stress (2,3). It is generally accepted that lipid rearrangements [...]

Short carbon nanotubes spontaneously insert into lipid bilayers and live cell membranes to form channels with useful and tunable transport properties that make them a promising biomimetic nanopore platform for developing cell interfaces, studying nanofluidic transport in biological channels, and creating stochastic sensors. Carbon-nanotube porins (Noy MH) Synthetic analogues of biological membrane channels that match the latter's high efficiency and exquisite selectivity for transporting ions and molecules could find many applications. Although it is possible to produce nanopores of a size comparable to that of protein channels, replicating their affinity and transport properties remains challenging. Jia Geng et al. now show that short (10-nm-long) single-wall carbon nanotubes spontaneously insert into lipid bilayers and live cell membranes to form channels with useful and tuneable transport properties. These carbon-nanotube channel-forming molecules or porins offer a promising biomimetic nanopore platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors. There is much interest in developing synthetic analogues of biological membrane channels.sup.1 with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up.sup.2 and top-down.sup.3 methods can produce nanopores of a size comparable to that of endogenous protein channels, but replicating their affinity and transport properties remains challenging. In principle, carbon nanotubes (CNTs) should be an ideal membrane channel platform: they exhibit excellent transport properties.sup.4,5,6,7,8 and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels.sup.1. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane.sup.9,10. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls.sup.11,12, and short tubes have been forced into membranes to create sensors.sup.13, yet membrane transport applications of short CNTs remain underexplored. Here we show that short CNTs spontaneously insert into lipid bilayers and live cell membranes to form channels that exhibit a unitary conductance of 70-100 picosiemens under physiological conditions. Despite their structural simplicity, these 'CNT porins' transport water, protons, small ions and DNA, stochastically switch between metastable conductance substates, and display characteristic macromolecule-induced ionic current blockades. We also show that local channel and membrane charges can control the conductance and ion selectivity of the CNT porins, thereby establishing these nanopores as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors., Author(s): Jia Geng [sup.1] [sup.2] [sup.3] , Kyunghoon Kim [sup.1] [sup.3] [sup.4] , Jianfei Zhang [sup.2] , Artur Escalada [sup.5] , Ramya Tunuguntla [sup.1] [sup.3] , Luis R. Comolli [sup.6] [...]

The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial fission, but the regulatory mechanisms remain ambiguous. Here we found that a conserved, intrinsically disordered, six-residue S hort Li near M otif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo . Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, leading to the fission of model membranes in vitro . In vivo , the availability of the native CT-SLiM is a requirement for productive mitochondrial fission, as both non-native extension and deletion of the CT-SLiM severely impair its progression. Thus, contrary to prevailing models, Drp1-catalyzed mitochondrial fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics., Competing Interests: Additional Declarations: Competing interests: The authors declare no competing interests.

Membrane fusion and fission are indispensable parts of intracellular membrane recycling and transport. Electrophysiological techniques have been instrumental in discovering and studying fusion and fission pores, the key intermediates shared by both processes. In cells, electrical admittance measurements are used to assess in real time the dynamics of the pore conductance, reflecting the nanoscale transformations of the pore, simultaneously with membrane leakage. Here, we described how this technique is adapted to in vitro mechanistic analyses of membrane fission by dynamin 1 (Dyn1), the protein orchestrating membrane fission in endocytosis. We reconstitute the fission reaction using purified Dyn1 and biomimetic lipid membrane nanotubes of defined geometry. We provide a comprehensive protocol describing simultaneous measurements of the ionic conductance through the nanotube lumen and across the nanotube wall, enabling spatiotemporal correlation between the nanotube constriction by Dyn1, leading to fission and membrane leakage. We present examples of "leaky" and "tight" fission reactions, specify the resolution limits of our method, and discuss how our results support the hemi-fission conjecture.

Morphological plasticity of biological membrane is critical for cellular life, as cells need to quickly rearrange their membranes. Yet, these rearrangements are constrained in two ways. First, membrane transformations may not lead to undesirable mixing of, or leakage from, the participating cellular compartments. Second, membrane systems should be metastable at large length scales, ensuring the correct function of the particular organelle and its turnover during cellular division. Lipids, through their ability to exist with many shapes (polymorphism), provide an adequate construction material for cellular membranes. They can self-assemble into shells that are very flexible, albeit hardly stretchable, which allows for their far-reaching morphological and topological behaviors. In this article, we will discuss the importance of lipid polymorphisms in the shaping of membranes and its role in controlling cellular membrane morphology.