Florian Wollweber

The emergence of eukaryotes from Asgard archaeal ancestors ^1–5 likely required a specialized cell envelope that enabled sustained physical and metabolic interactions with proto-mitochondrial symbionts ^6–10 . While the protrusion-rich cell architecture of Asgard archaea may enable such interactions, the molecular composition of their cell surface and its role in establishing cell-cell contacts remains unknown. Here, we developed a “visual surfaceomics” approach, integrating subtomogram averaging, predictive structural modeling, transcriptomics, proteomics, and expansion microscopy to identify surface proteins in situ . We show that Asgard cell bodies and protrusions lack an archaea-typical paracrystalline S-layer and are instead decorated by two main classes of surface proteins: subtilisin-like serine proteases (SubSs) and oligopeptide-binding proteins (OppAs). SubSs were detected as membrane-anchored dimers and were sparsely distributed across the surface. OppAs showed near-total membrane coverage but lacked any rigid, higher order assembly. Thermal shift assays and single particle cryo-EM showed that OppA binds oligopeptides in vitro . Comparative genomics, transcriptomics, and proteomics suggest that this peptidophilic surface architecture is conserved across Asgard archaeal phyla. Finally, we discovered that the cell surface was remodeled at cell-cell contact sites. Altogether, our integrative approach revealed a metabolically specialized and structurally flexible cell surface that couples extracellular protein degradation with peptide capture and uptake, while potentially contributing to establishing and maintaining the symbiotic interactions of Asgard archaea.

SUMMARY: Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a “Filament IDentification” (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of gently lysed cells. FilamentID identified the mitochondrial filaments as the conserved aldehyde dehydrogenase Ald4 ^ALDH2 and the nucleoplasmic/cytoplasmic filaments being composed of acetyl-CoA synthetase Acs1 ^ACSS2 . The near-native high-resolution structures revealed the mechanism underlying polymerization and enabled us to perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores, and more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts. HIGHLIGHTS: FilamentID: a multiscale imaging workflow to characterize cellular filaments of unknown composition The conserved aldehyde dehydrogenase Ald4 ^ALDH2 polymerizes into filament arrays within meiotic mitochondria The conserved acetyl-CoA synthetase Acs1 ^ACSS2 forms filament arrays in the nucleus and the cytoplasm Metabolites mediate Acs1 polymerization to store Acs1 in an inactive state in gametes and starved cells Acs1 filament formation is required for efficient return to growth from starvation stress

Tc toxins are virulence factors of bacterial pathogens. Although their structure and intoxication mechanism are well understood, it remains elusive where this large macromolecular complex is assembled and how it is released. Here we show by an integrative multiscale imaging approach that Yersinia entomophaga Tc (YenTc) toxin components are expressed only in a subpopulation of cells that are ‘primed’ with several other potential virulence factors, including filaments of the protease M66/StcE. A phage-like lysis cassette is required for YenTc release; however, before resulting in complete cell lysis, the lysis cassette generates intermediate ‘ghost’ cells, which may serve as assembly compartments and become packed with assembled YenTc holotoxins. We hypothesize that this stepwise mechanism evolved to minimize the number of cells that need to be killed. The occurrence of similar lysis cassettes in diverse organisms indicates a conserved mechanism for Tc toxin release that may apply to other extracellular macromolecular machines.

Asgard archaea are considered to be the closest known relatives of eukaryotes. Their genomes contain hundreds of eukaryotic signature proteins (ESPs), which inspired hypotheses on the evolution of the eukaryotic cell^1–3. A role of ESPs in the formation of an elaborate cytoskeleton and complex cellular structures has been postulated^4–6, but never visualized. Here we describe a highly enriched culture of ‘CandidatusLokiarchaeum ossiferum’, a member of the Asgard phylum, which thrives anaerobically at 20 °C on organic carbon sources. It divides every 7–14 days, reaches cell densities of up to 5 × 10⁷cells per ml and has a significantly larger genome compared with the single previously cultivated Asgard strain⁷. ESPs represent 5% of its protein-coding genes, including four actin homologues. We imaged the enrichment culture using cryo-electron tomography, identifying ‘Ca. L. ossiferum’ cells on the basis of characteristic expansion segments of their ribosomes. Cells exhibited coccoid cell bodies and a network of branched protrusions with frequent constrictions. The cell envelope consists of a single membrane and complex surface structures. A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions. The twisted double-stranded architecture of the filaments is consistent with F-actin. Immunostaining indicates that the filaments comprise Lokiactin—one of the most highly conserved ESPs in Asgard archaea. We propose that a complex actin-based cytoskeleton predated the emergence of the first eukaryotes and was a crucial feature in the evolution of the Asgard phylum by scaffolding elaborate cellular structures.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie–shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function.

Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.

Balanced fusion and fission are key for the proper function and physiology of mitochondria^1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals^3,4,5. Mgm1 is required for the preservation of mitochondrial DNA in yeast⁶, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy—a genetic disorder that affects the optic nerve^7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm1^9,10 or mammalian cells that lack OPA1 display fragmented mitochondria^11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane—not the inner membrane—fuses in the absence of functional Mgm1¹³. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture^10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions¹⁵. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission¹⁶. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.