Biology subjects

Ganeva, I.

Publications and source records attributed to Ganeva, I..

5 recordsLinked to original sources

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

Large transient assemblies of Apaf1 constitute the apoptosome in cells

Upon cell death signals, the apoptotic protease-activating factor Apaf1 and cytochrome c interact to form the apoptosome complex. The apoptosome is crucial for mitochondrial apoptosis, as it activates caspases that dismantle the cell. However, the assembly mechanism and appearance of the apoptosome in vivo remain unclear. We show that upon onset of apoptosis, Apaf1 molecules accumulate into multiple foci per cell. Disassembly of the foci is linked to survival of the cell. Structurally, Apaf1 foci resemble organelle-sized, cloud-like assemblies. Foci form upon specific molecular interactions with cytochrome c and depending on procaspase-9. We propose that Apaf1 foci correspond to the apoptosome in cells. Transientness and ultrastructure of Apaf1 foci suggest that the dynamic spatiotemporal organisation of apoptosome components regulates progression of apoptosis.

cell biology↗

bioRxiv2021

The architecture of Cidec-mediated interfaces between lipid droplets

Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously (1). In white adipocytes, they reach up to 100 {micro}m in size, occupying >90% of the cell. Cidec, which is strictly required for the formation of such large LDs, is concentrated at interfaces between adjacent LDs and facilitates the directional flux of neutral lipids from the smaller to the larger LD (2-7). However, the mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs has remained elusive. Here we visualised interfaces between LDs by electron cryo-tomography (cryo-ET) and analysed the kinetics of lipid transfer by quantitative live fluorescence microscopy (FM). We show that transfer occurs through closely apposed intact monolayers, is slowed down by increasing the distance between the monolayers and follows exponential kinetics suggesting a pressure-driven mechanism. We thus propose that unique architectural features of LD-LD interfaces are mechanistic determinants of neutral lipid transfer.

cell biology↗

bioRxiv2020

The supramolecular landscape of growing human axons

During brain development, human axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly inaccessible to imaging at molecular resolution in a near-native context. Here we apply cryo-correlative light microscopy and electron tomography to growing axonal tracts from human cerebral organoids. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These data provide an unprecedented resource and reveal a molecular architecture that helps explain the unique biology of growing human axons.

cell biology↗

bioRxiv2018

Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis

During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher-order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures.

cell biology↗