Biology subjects

Sachl, R.

Publications and source records attributed to Sachl, R..

3 recordsLinked to original sources

bioRxiv2024

Quantitative imaging of species-specific lipid transport in mammalian cells

Eukaryotic cells produce over 1000 different lipid species which tune organelle membrane properties, control signalling and store energy1,2. How lipid species are selectively sorted between organelles to maintain specific membrane identities is largely unknown due to the difficulty to image lipid transport in cells3. Here, we measured transport and metabolism of individual lipid species in mammalian cells using time-resolved fluorescence imaging of bifunctional lipid probes in combination with ultra-high resolution mass spectrometry and mathematical modelling. Quantification of lipid flux between organelles revealed that directional, non-vesicular lipid transport is responsible for fast, species-selective lipid sorting compared to slow, unspecific vesicular membrane trafficking. Using genetic perturbations, we found that coupling between active lipid flipping and passive non-vesicular transport is a mechanism for directional lipid transport. Comparison of metabolic conversion and transport rates showed that non-vesicular transport dominates the organelle distribution of lipids while species-specific phospholipid metabolism controls neutral lipid accumulation. Our results provide the first quantitative map of retrograde lipid flux in cells4. We anticipate that our pipeline for quantitative mapping of lipid flux through physical and chemical space in cells will boost our understanding of lipids in cell biology and disease.

cell biology↗

bioRxiv2023

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space

Fibroblast Growth Factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by PI(4,5)P2-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the protein-protein interaction interface through which FGF2 binds to the 1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we provide insights into a FGF2 dimerization interface that brings C95 residues in close proximity, resulting in disulfide bridged FGF2 dimers. We propose a mechanism by which they bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95 bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

biochemistry↗

bioRxiv2022

Inter-leaflet Organization of Membrane Nanodomains: What Can(not) Be Resolved by FRET?

Plasma membranes as well as their simplified model systems show an inherent nanoscale heterogeneity. As a result of strong interleaflet interactions, these nanoheterogeneities (called here lipid nanodomains) can be found in perfect registration (i.e. nanodomains in the inner leaflet are registered with the nanodomains in the outer leaflet). Alternatively, they might be inter-leaflet independent, anti-registered or located asymmetrically in one bilayer leaflet only. To distinguish these scenarios from each other appears to be an experimental challenge. In this work, we analyzed the potential of Forster resonance energy transfer (FRET) to characterize inter-leaflet organization of nanodomains. We generated in-silico time-resolved fluorescence decays for a large set of virtual as well as real donor/acceptor pairs distributed over the bilayer containing registered, independent, anti-registered or asymmetrically distributed nanodomains. In this way, we were able to identify conditions that gave satisfactory or unsatisfactory resolution. Overall, FRET appears as a robust method that - when using D/A pairs with good characteristics - yields otherwise difficult-to-reach characteristics of membrane lipid nanodomains. STATEMENT OF SIGNIFICANCEThis work first explores the potential of Forster resonance energy transfer (FRET) to characterize inter-leaflet nanodomain coupling and then shows how a FRET experiment can designed to achieve optimal resolution towards nanodomain coupling. Importantly, the analysis identifies as the most critical the following parameters fundamentally affecting the resolution of FRET: the Forster radius and its value related to the inter-layer distance at which donors and acceptors in the opposing membrane leaflets are separated from each other and the donor and acceptor partition coefficients characterizing their distribution between the domain and nondomain region. By setting these parameters correctly, FRET allows for the characterization of inter-leaflet nanodomain organization with unprecedented detail.

biophysics↗