Topological transitions of lipid membranes are ubiquitous in key biological processes for cell life, like neurotransmission, fertilization, morphogenesis, and viral infections. Despite this, they are not well understood due to their multiscale nature, which limits the use of molecular models and calls for a mesoscopic approach such as the celebrated Canham-Helfrich one. Unfortunately, such a model cannot handle topological transitions, hiding the crucial involved forces and the appearance of the experimentally observed hemifused intermediates. In this work, we describe the membrane as a diffuse interface preserving the Canham-Helfrich elasticity. We show that pivotal features of the hemifusion pathway are captured by this mesoscopic approach, e.g. a (meta)stable hemifusion state and the fusogenic behavior of negative monolayer spontaneous curvatures. The membrane lateral stress profile is calculated as a function of the elastic rigidities, yielding a coarse-grained version of molecular models findings. Insights into the fusogenic mechanism are reported and discussed.

The Coat Protein I (COPI) complex forms vesicles from Golgi membrane for retrograde transport among the Golgi stacks, and also from the Golgi to the endoplasmic reticulum (ER). We have been elucidating the mechanistic details of COPI vesicle formation through a reconstitution system that involves the incubation of Golgi membrane with purified components. This approach has enabled us recently to gain new insight into how certain lipids are critical for the fission stage of COPI vesicle formation. Lipid geometry has been proposed to act in the formation of transport carriers by promoting membrane curvature. However, evidence for this role has come from studies using simplified membranes, while confirmation in the more physiologic setting of native membranes has been challenging, as such membranes contain a complex composition of lipids and proteins. We have recently refined the COPI reconstitution system to overcome this experimental obstacle. This has led us to identify an unanticipated type of lipid geometry needed for COPI vesicle fission. This chapter describes the approach that we have developed to enable this discovery. The methodologies include: (i) preparation Golgi membrane from cells that are deficient in a particular lipid enzyme activity and (ii) functional rescue of this deficiency by introducing the product of the lipid enzyme, with experiments being performed at the in vitro level to gain mechanistic clarity and at the in vivo level to confirm physiologic relevance.

Heterozygous pathogenic variants in DNM1 cause developmental and epileptic encephalopathy (DEE) as a result of a dominant-negative mechanism impeding vesicular fission. Thus far, pathogenic variants in DNM1 have been studied with a canonical transcript that includes the alternatively spliced exon 10b. However, after performing RNA sequencing in 39 pediatric brain samples, we find the primary transcript expressed in the brain includes the downstream exon 10a instead. Using this information, we evaluated genotype-phenotype correlations of variants affecting exon 10a and identified a cohort of eleven previously unreported individuals. Eight individuals harbor a recurrent de novo splice site variant, c.1197-8G>A (GenBank: NM_001288739.1), which affects exon 10a and leads to DEE consistent with the classical DNM1 phenotype. We find this splice site variant leads to disease through an unexpected dominant-negative mechanism. Functional testing reveals an in-frame upstream splice acceptor causing insertion of two amino acids predicted to impair oligomerization-dependent activity. This is supported by neuropathological samples showing accumulation of enlarged synaptic vesicles adherent to the plasma membrane consistent with impaired vesicular fission. Two additional individuals with missense variants affecting exon 10a, p.Arg399Trp and p.Gly401Asp, had a similar DEE phenotype. In contrast, one individual with a missense variant affecting exon 10b, p.Pro405Leu, which is less expressed in the brain, had a correspondingly less severe presentation. Thus, we implicate variants affecting exon 10a as causing the severe DEE typically associated with DNM1-related disorders. We highlight the importance of considering relevant isoforms for disease-causing variants as well as the possibility of splice site variants acting through a dominant-negative mechanism.

Eukaryotic cells internalize cargos specifically through clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). EndophilinA2 was shown as preferentially implicated in CIE, although initially involved in CME. Here, we investigated the native interplay of endophilinA2 and dynamin2 during CME as compared to CIE. We developed an unbiased integrative approach based on genome engineering, robust tracking methodology, and advanced analytics. We statistically identified CME and CIE subpopulations corresponding to abortive, active, and static endocytic events. Depletion of dynamin2 strongly affected active CME and CIE events, whereas the absence of endophilinA2 impacted only CIE. Accordingly, we demonstrated that endophilinA2 is needed for dynamin2 recruitment during CIE, but not in CME. Despite these differences, endophilinA2 and dynamin2 acted at the latest stage of endocytosis within a similar stoichiometry in both mechanisms. Thus, we propose a conserved function of dynamin2 and endophilinA2 in vesicle scission, but a differential regulation of their recruitment during CME and CIE.