Biology subjects

Moeller, A.

Publications and source records attributed to Moeller, A..

25 records · Page 2Linked to original sources

bioRxiv2023

Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF-activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro, and a shift from Rab5 to more Rab7 positive structures in Drosophila nephrocytes and yeast, suggesting faster endosomal maturation. Using modeling, we further identify a conserved Rab5 binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Rab-conversion and organelle maturation along the endomembrane system. SummaryTransport of proteins via the endolysosomal pathway requires Rab5 on early endosomes, which is replaced by Rab7 on late endosomes. Here, we identify distinct regulatory sites in the Rab7 activator, the Mon1-Ccz1 complex, shedding light on the regulation of Rab5 to Rab7 transition.

cell biology↗

bioRxiv2023

Structure of the ceramide-bound SPOTS complex

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyl-transferase (SPT) catalyzes the rate limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.

biochemistry↗

bioRxiv2022

Structure of the lysosomal membrane fusion machinery

Lysosomes are of central importance in cellular recycling, nutrient signaling 1,2 and endocytosis, and are tightly connected to autophagy 3 and the invasion of pathogenic bacteria and viruses 1,4. Lysosomal fusion events are fundamental to cell survival and require HOPS, a conserved heterohexameric tethering complex 5,6. HOPS recognizes and binds small membrane-associated GTPases on lysosomes and organelles, and assembles membrane bound SNAREs for fusion 7,8. Through tethering, HOPS brings membranes in close proximity to each other and significantly increases fusion efficacy by catalysing SNARE assembly. Consequently, different HOPS mutations are causative for severe diseases 6. Despite its fundamental cellular duties, it remained speculative how HOPS fulfils its function as high-resolution structural data were unavailable. Here, we used cryo-electron microscopy to reveal the structure of HOPS. In the complex, two central subunits form the backbone and an assembly hub for the functional domains. Two GTPase binding units extend to opposing ends, while the SNARE binding module points to the side, resulting in a triangular shape of the complex. Unlike previously reported, HOPS is surprisingly rigid and extensive flexibility is confined to its extremities. We show that HOPS complex variants with mutations proximal to the backbone can still tether membranes but fail to efficiently promote fusion indicating, that the observed integrity of HOPS is essential to its function. In our model, the core of HOPS acts as a counter bearing between the flexible GTPase binding domains. This positions the SNARE binding module exactly between the GTPase anchored membranes to promote fusion. Our structural and functional analysis reveals the link between the spectacular architecture of HOPS and its mechanism that couples membrane tethering and SNARE assembly, to catalyse lysosomal fusion.

biophysics↗

bioRxiv2022

Mechanism of multimodal substrate translocation in P-glycoprotein

P-glycoprotein (Pgp) is a prototypical ABC transporter of great biological and clinical significance that confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs1-3. Decades of structural and biochemical studies have provided insights into how Pgp binds diverse compounds4-9, but how they are translocated through the membrane has remained elusive. Here, we covalently attached a cyclic substrate to discrete sites of Pgp and determined multiple complex structures in inward- and outward-facing states by cryoEM. In conjunction with molecular dynamics simulations, our structures trace the substrate passage across the membrane and identify conformational changes in transmembrane helix 1 (TM1) as regulators of substrate transport. In mid-transport conformations, TM1 breaks at glycine 72. Mutation of this residue significantly impairs drug transport of Pgp in vivo, corroborating the importance of its regulatory role. Importantly, our data suggest that the cyclic substrate can exit Pgp without the requirement of a wide-open outward-facing conformation, diverting from the common efflux model for Pgp and other ABC exporters. The substrate transport mechanism of Pgp revealed here pinpoints critical targets for future drug discovery studies of this medically relevant system.

biophysics↗

bioRxiv2022

Spatial and social structure of rewilded laboratory mice

Social behavior and social organization have major influences on individual health and fitness. Yet, biomedical research focuses on studying a few genotypes under impoverished social conditions. Understanding how lab conditions have modified social organizations of model organisms, such as lab mice, relative to natural populations is a missing link between socioecology and biomedical science. Using a common garden design, we describe the formation of social structure in the well-studied laboratory mouse strain, C57BL/6J, in replicated mixed-sex populations over 10-day trials compared to control trials with wild-derived outbred house mice in outdoor field enclosures. We focus on three key features of mouse social systems: (i) territory establishment in males, (ii) female social relationships, and (iii) the social networks formed by the populations. Male territorial behaviors were similar but muted in C57 compared to wild-derived mice. Female C57 sharply differed from wild-derived females, showing little social bias toward cage mates and exploring substantially more of the enclosures compared to all other groups. Female behavior consistently generated denser social networks in C57 than in wild-derived mice. The repeatable societies formed under field conditions highlights opportunities to experimentally study the interplay between society and individual biology using model organisms.

animal behavior and cognition↗

bioRxiv2019

Stabilization of α-synuclein oligomers using formaldehyde

The group of neurodegenerative diseases, Parkinsons disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type -synuclein (-syn) aggregates within degenerating brain cells. -syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating -syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized -syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific -syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked -syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked -syn oligomers could serve as important calibrators to facilitate comparative and standardized -syn biomarker studies going forward.

neuroscience↗

bioRxiv2019

Structure and autoregulation of a P4-ATPase lipid flippase

P4-ATPases are lipid flippases that drive active transport of phospholipids from the exoplasmic or lumenal to the cytosolic leaflets of eukaryotic membranes to maintain their asymmetric lipid composition. The molecular architecture of P4-ATPases and how they work in lipid recognition and transport has remained elusive. Using cryo-electron microscopy we have determined the structures of a P4-ATPase, specifically of the Saccharomyces cerevisiae Drs2p-Cdc50p, which is a phosphatidylserine and phosphatidylethanolamine specific lipid flippase. Drs2p-Cdc50p is autoinhibited by the Drs2p C-terminal tail and activated by phosphatidylinositol-4 phosphate (PI4P). We present three structures representing an autoinhibited, an intermediate, and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of auto-inhibition and PI4P-dependent activation. We observe the opening of a putative flippase pathway engaging conserved residues Ile508 of transmembrane segment 4 and Lys1018 and polar residues of transmembrane segment 5 in the centre of the lipid bilayer.

molecular biology↗