Juan Manuel Iglesias‐Artola

Clathrin-mediated endocytosis is a major transport route for proteins from the plasma membrane to the interior of the cell. While the recruitment of cargo proteins to clathrin-coated pits is well understood, it remains an open question whether lipids are also sorted by this process. Here, to address this question, we combined super-resolution stimulated emission depletion imaging of bifunctional lipid probes with mathematical modelling. Quantification of ten different lipid species revealed significant differences in pit partitioning, ranging from slight enrichment to moderate exclusion. We find that the lipid asymmetry in the plasma membrane is sufficient to explain the observed trend. Taken together, our findings imply that clathrin-mediated endocytosis has a minor selectivity for cytoplasmic leaflet lipids, but overall does not substantially contribute to lipid sorting compared with non-vesicular trafficking.

Electrochemical gradients are essential to the functioning of cells and form across membranes using active transporters. Here we show in contrast that condensed biomolecular systems—often termed condensates—sustain pH gradients without any external energy input. By studying individual condensates on the micrometre scale using a microdroplet platform, we reveal dense-phase pH shifts towards conditions of minimal electrostatic repulsion. We demonstrate that protein condensates can drive substantial alkaline and acidic gradients, which are compositionally tunable and can extend to complex architectures sustaining multiple unique pH conditions simultaneously. Through in silico characterization of human proteomic condensate networks, we further highlight potential wide-ranging electrochemical properties emerging from condensation in nature, while correlating intracellular condensate pH gradients with complex biomolecular composition. Together, the emergent nature of condensation shapes distinct pH microenvironments, thereby creating a regulatory mechanism to modulate biochemical activity in living and artificial systems.

Zusammenfassung: Ether‐Glycerophospholipide enthalten einen langkettigen Alkohol, der über eine Alkyl‐ oder Vinyletherbindung an der sn1 ‐Position des Glycerin‐Grundgerüsts gebunden ist. Etherlipide sind für zelluläre Physiologie und menschliche Gesundheit hoch relevant. Ihre Zellbiologie ist jedoch weitgehend unklar, da es an Methoden zur Analyse ihrer subzellulären Lokalisierung und Identifikation interagierender Proteine fehlt. Als Ausgangspunkt zellbiologischer Studien wurden bifunktionelle Etherlipid‐Sonden mit Diazirin‐ und Alkin‐Gruppen synthetisiert. Um die Kinetik des intrazellulären Etherlipid‐Transports in Säugetierzellen zu untersuchen, wurde eine Kombination aus Fluoreszenzbildgebung, KI‐unterstützter Bildanalyse und mathematischer Modellierung verwendet. Wir stellen fest, dass alkylverknüpfte Etherlipide bis zu zweifach schneller transportiert werden als vinylverknüpfte Spezies (Plasmalogene). Dies deutet darauf hin, dass die zelluläre Lipidtransportmaschinerie in der Lage ist, zwischen Verknüpfungstypen zu unterscheiden, die sich nur durch zwei Wasserstoffatome unterscheiden. Der Transport von Etherlipiden erfolgt dabei überwiegend über nichtvesikuläre Mechanismen, wobei der Beitrag des vesikulären Transports je nach Zelltyp variiert. Insgesamt deuten unsere Ergebnisse darauf hin, dass die unterschiedliche Erkennung von Alkyl‐ und Vinyletherlipiden durch Lipidtransferproteine zu deren unterschiedlichen biologischen Funktionen beiträgt. Die hier vorgestellten Sonden werden es in Zukunft ermöglichen, die Biologie der Etherlipide genauer zu untersuchen, z.B. durch die Identifizierung der interagierenden Proteine.

Ether glycerophospholipids bear a long chain alcohol attached via an alkyl or vinyl ether bond at the sn1 position of the glycerol backbone. Ether lipids play a significant role in physiology and human health. However, their cellular functions remain largely unknown due to a lack of tools for identifying their subcellular localization and interacting proteins. Here, we address this methodological gap by synthesizing minimally modified bifunctional ether lipid probes by introducing diazirine and alkyne groups. To interrogate the subcellular kinetics of intracellular ether lipid transport in mammalian cells, we used a combination of fluorescence imaging, machine learning‐assisted image analysis, and mathematical modelling. We find that alkyl‐linked ether lipids are transported up to twofold faster than vinyl‐linked species (plasmalogens), pointing to yet undiscovered cellular lipid transport machinery able to distinguish between linkage types differing by as little as two hydrogen atoms. We find that ether lipid transport predominantly occurs via non‐vesicular pathways, with varying contributions from vesicular mechanisms between cell types. Altogether, our results suggest that differential recognition of alkyl‐ and vinyl ether lipids by lipid transfer proteins contributes to their distinct biological functions. In the future, the probes reported here will enable studying ether lipid biology in much greater detail through identification of interacting proteins and in‐depth characterization of intracellular ether lipid dynamics.

Eukaryotic cells produce over 1,000 different lipid species that tune organelle membrane properties, control signalling and store energy^1,2. How lipid species are selectively sorted between organelles to maintain specific membrane identities is largely unclear, owing to the difficulty of imaging lipid transport in cells³. Here we measured the retrograde 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, in contrast to the slow, unspecific vesicular membrane trafficking. Using genetic perturbations, we found that coupling between energy-dependent lipid flipping and 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 mapping of lipid flux through physical and chemical space in cells will boost our understanding of lipids in cell biology and disease.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis, are often associated with mutations in stress granule proteins. Aberrant stress granule condensate formation is associated with disease, making it a potential target for pharmacological intervention. Here, we identified lipoamide, a small molecule that specifically prevents cytoplasmic condensation of stress granule proteins. Thermal proteome profiling showed that lipoamide stabilizes intrinsically disordered domain-containing proteins, including SRSF1 and SFPQ, which are stress granule proteins necessary for lipoamide activity. SFPQ has redox-state-specific condensate dissolving behavior, which is modulated by the redox-active lipoamide dithiolane ring. In animals, lipoamide ameliorates aging-associated aggregation of a stress granule reporter protein, improves neuronal morphology and recovers motor defects caused by amyotrophic lateral sclerosis-associated FUS and TDP-43 mutants. Thus, lipoamide is a well-tolerated small-molecule modulator of stress granule condensation, and dissection of its molecular mechanism identified a cellular pathway for redox regulation of stress granule formation.

Clathrin-mediated endocytosis is a major transport route for proteins from the plasma membrane to the interior of the cell. While the recruitment of cargo proteins to clathrin-coated pits is well understood, it remains an open question if lipids are also sorted by this process. To address this question, we combined super-resolution STED imaging of bifunctional lipid probes with mathematical modeling. Quantification of 10 different lipid species revealed significant differences in pit partitioning, ranging from slight enrichment to moderate exclusion. We find that the lipid asymmetry in the plasma membrane is sufficient to explain the observed trend. Taken together, our findings imply that clathrin-mediated endocytosis has a minor selectivity for cytoplasmic leaflet lipids, but overall does not significantly contribute to lipid sorting compared to non-vesicular trafficking. More broadly, we believe that lipid super-resolution imaging will be a powerful approach to quantify lipid partitioning into membrane structures in cells.

Protein phase separation has become a widely studied phenomena in biology with implications in cell metabolism and disease. The study of phase separating proteins often relies on the precise determination of their phase diagrams. These phase diagrams give information on the protein concentration required for condensate formation and the respective concentration inside the condensate at defined external conditions (temperature, salt, pH). However, it has so far often proven difficult to accurately measure phase diagrams. Here, we report a method that is based on mass and volume conservation and defined reaction volumes, which we call inPhase. We can use this method to determine accurate values for both dilute and condensed branch protein concentrations. With this information we can produce accurate phase diagrams. We compare our method to the widely used quantitative fluorescence approach and find that it underestimates the partition factor into condensates at least two-fold for FUS and PGL-3. The accessibility of our method opens the possibility for the thermodynamic assessment of entire protein families, generating sufficient quantitative data for testing theory, and for the screening of drugs in pharmaceutical research.

Ether glycerophospholipids bear a long chain alcohol attached via an alkyl or vinyl ether bond at the sn1 position of the glycerol backbone. Emerging evidence suggests that ether lipids play a significant role in physiology and human health but their precise cellular functions remain largely unknown. Here, we introduce bifunctional ether lipid probes bearing diazirine and alkyne groups to study ether lipid biology. To interrogate the kinetics of intracellular ether lipid transport in mammalian cells we used a combination of fluorescence imaging, machine learning-assisted image analysis and mathematical modelling. We find that alkyl-linked ether lipids are transported up to twofold faster than vinyl-linked plasmalogens, suggesting that the lipid transport machinery can distinguish between linkage types differing by as little as two hydrogen atoms. We find that ether lipid transport predominantly occurs via non-vesicular pathways, with varying contributions from vesicular mechanisms between cell types. Altogether, our results suggest that differential recognition of alkyl- and vinyl ether lipids by lipid transfer proteins contributes to their distinct biological functions. In the future, the probes reported here will enable studying ether lipid biology in much greater detail through identification of interacting proteins and in-depth characterization of intracellular ether lipid dynamics.

The study of biological processes involving live microscopy techniques requires adequate temperature control to respect the physiology of the organism under study. We present here a design strategy for a microscope temperature stage based on thermoelectric elements. The design allows the user to access a range of temperatures below and above room temperature and can accommodate samples of different geometries. In addition, by cooling simultaneously the sample insert and the objective, we minimize the temperature gradients along the sample for large magnification objectives requiring immersion oil. We illustrate how this design can be used to study the physiology of the zebrafish embryo over the temperature tolerance of this species. We envision that this device could benefit the communities using model and non-model organisms with physiological temperatures different from typical mammalian cell culture incubation in biomedical research.