Biology subjects

Espadas, J.

Publications and source records attributed to Espadas, J..

6 recordsLinked to original sources

bioRxiv2025

Molecular basis for cellular compartmentalization by an ancient membrane fission mechanism

The emergence of cell compartmentalization depends on membrane fission to create the endomembrane compartments. In eukaryotes, membrane fission is commonly executed by ESCRT-III, a protein complex conserved in all domains of life. However, whether membrane fission was an ancestral ESCRT-III activity predating eukaryogenesis remains unknown. Here we show that ESCRT-IIIA from Asgard Heimdallarchaeota, the closest archaeal relatives of eukaryotes, performs membrane fission through an N-terminal amphipathic helix, which we term Hofund. In eukaryotes, Hofund is fragmented across ESCRT-IIIA paralogs, and disrupting these regions causes severe fission defect in yeast. Remarkably, Heimdallarchaeota Hofund restores fission when fused to defective eukaryotic paralogs. These findings suggest that ESCRT-III-mediated fission arose before eukaryogenesis and later diversified to support the regulatory complexity of eukaryotic compartmentalization.

cell biology↗

bioRxiv2024

Two-dimensional condensates of HRS drive the assembly of flat clathrin lattices on endosomes

In cells, the curved clathrin structures in vesicle budding are well characterized, while the flat ones remain poorly understood. We reconstituted the flat assembly of ESCRT-0 protein HRS and clathrin onto lipid membranes in vitro. HRS was found to form protein condensates. These condensates spread as a two-dimensional layer on negatively charged membranes and promoted the assembly of clathrin into a flat coat. Correlative cryo-tomography of HRS-labeled endosomes revealed a pure hexagonal lattice, consistent with flat clathrin structures. Cholesterol enhanced HRS recruitment to the membrane both in cells and in supported bilayers. Furthermore, cholesterol promoted the phase separation of HRS onto membranes, which in turn concentrated cholesterol underneath. This positive feedback promoted the formation of HRS-clathrin microdomains that sorted reconstituted ubiquitinated cargoes. Altogether, our results show that the unique architecture of ESCRT-0 is assembled by the two-dimensional phase-separation of HRS which drives the assembly of flat clathrin coats.

biochemistry↗

bioRxiv2024

Evolutionarily conserved principles of ESCRT-III-mediated membrane remodelling revealed by a two-subunit Asgard archaeal system

ESCRT-III proteins assemble into composite polymers that undergo stepwise changes in composition and structure to deform membranes across the tree of life. Here, using a phylogenetic analysis we demonstrate that the two ESCRT-III proteins present in our closest archaeal relatives are evolutionarily related to B-type and A-type eukaryotic paralogues, which initiate and execute membrane remodelling, respectively. This deep homology is reflected in ESCRT-III structure and function as demonstrated by the fact that ESCRT-IIIB assembles into parallel arrays on planar membranes to initiate membrane deformation, and is required to recruit ESCRT-IIIA to generate composite polymers. ESCRT-IIIA homopolymers can then remodel membranes into tubes, as a likely prelude to scission. Taken together, this analysis reveals a set of conserved principles governing ESCRT-III-dependent membrane remodelling that first emerged with the evolution of a two-component ESCRT-III system in the Asgard archaea, and which continue to underlie complex multi-component, ESCRT-III-dependent membrane remodelling in eukaryotes.

evolutionary biology↗

bioRxiv2023

Mechanism for Vipp1 spiral formation, ring biogenesis and membrane repair

ESCRT-III family proteins build dynamic filaments that remodel membrane. Although the transition of planar filaments into 3D membrane budding structures is fundamental for their function, the geometric changes in polymer architecture driving the transition remain obscure. Here we show how bacterial Vipp1 polymerises into dynamic planar sheets and spirals on membrane. The spirals converge to form a central ring like those known to bud membrane. To probe how Vipp1 morphs between polymers, we determine the architecture of multiple helical filaments. As well as describing filament constriction and membrane tubulation, the geometric relationship between helical and planar lattices enables Vipp1 sheets and spirals to be modelled. Moreover, the helical structures show filaments twisting - a process needed for Vipp1 to transition between planar and 3D architectures. Given the structural conservation between Vipp1 and ESCRT-III, our results may represent the broad changes in geometry required for some ESCRT-III filaments to switch between 2D and 3D forms.

microbiology↗

bioRxiv2021

GTP and lipids control self-assembly and functional promiscuity of Dynamin2 molecular machinery

Dynamin2 GTPase (Dyn2) is a crucial player in clathrin-mediated endocytosis. Dyn2 is tetrameric in cytoplasm and self-assembles into functional units upon membrane binding. How the curvature activities and functionality of Dyn2 emerge during self-assembly and are regulated by lipids remains unknown. Here we reconstituted the Dyn2 self-assembly process using membrane nanotubes (NT) and vesicles and characterized it using single- molecule fluorescence microscopy, optical tweezers force spectroscopy and cryo-electron microscopy. On NTs, Dyn2 first forms small subhelical oligomers, which are already curvature active and display pronounced curvature sensing properties. Conical lipids and GTP promote their further self-assembly into helical machinery mediating the NT scission. In the presence of large unilamellar vesicles (LUVs), an alternative self- assembly pathway emerges where the subhelical oligomers form membrane tethering complexes mediating LUV-NT binding. Reconstitution of tethering in the LUV system revealed that lipid mixing is controlled by conical lipid species, divalents, GTP, and SH3 binding partners of Dyn2. In membranes with a high content of lipids with negative intrinsic curvature, cryo-EM revealed putative membrane contact sites made by Dyn2 clusters. On such membranes, with GTP lowered to 0.2 mM, both membrane fission and tethering activities become possible, indicating functional promiscuity of Dyn2. We conclude that GTP and lipids control both extent and topology of Dyn2 functional self-assembly. The function of Dyn2 oligomers evolves from curvature sensing, seen in subhelical Dyn2 oligomers, to curvature creation and fission, seen in Dyn2 helices. Under specific circumstances, such as downregulation of SH3 partners of Dyn2 and GTP depletion, membrane tethering activity can emerge in membrane systems enriched with conical lipids. Hence the Dyn2 functionality is actively adapted to lipidome, explaining its large habitat in the cells and tissues.

biophysics↗

bioRxiv2018

Dynamic constriction and fission of ER membranes by reticulon

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been widely associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. We found that the ER network has an embedded fragmentation mechanism based upon the ability of reticulons to produce fission of elongating network branches. In Drosophila, fission is counterbalanced by atlastin-driven fusion, with their imbalance leading to ER fragmentation. Live imaging of ER network dynamics upon ectopic expression of Drosophila reticulon linked fission to augmented membrane friction. Consistently, in vitro analysis revealed that purified reticulon produced velocity-dependent constriction and fission of lipid nanotubes pulled from a flat reservoir membrane. Fission occurred at elongation rates and pulling force ranges intrinsic to the ER network, thus suggesting a novel principle of organelle morphology regulation where the dynamic balance between fusion and fission is governed by membrane motility.

cell biology↗