Lennartz et al. evaluate lipid enrichment in clathrin-coated pits and observe that the differential lipid partitioning into clathrin-coated pits is largely driven by the lipid asymmetry of the plasma ...

Lennartz et al. evaluate lipid enrichment in clathrin-coated pits and observe that the differential lipid partitioning into clathrin-coated pits is largely driven by the lipid asymmetry of the plasma ...

Lennartz et al. evaluate lipid enrichment in clathrin-coated pits and observe that the differential lipid partitioning into clathrin-coated pits is largely driven by the lipid asymmetry of the plasma ...

Lennartz et al. evaluate lipid enrichment in clathrin-coated pits and observe that the differential lipid partitioning into clathrin-coated pits is largely driven by the lipid asymmetry of the plasma ...

To quantify the role of CME for sorting lipids at the plasma membrane, we optimized our recently developed lipid imaging workflow19 for super-resolution STED microscopy. The lipid imaging approach relies on near-native bifunctional lipids, which are modified with a diazirine and alkyne moiety. The diazirine can be photochemically cross-linked to neighbouring biomolecules, while the alkyne is used for labelling by click chemistry. These probes closely resemble native lipids with regard to their metabolism and biophysical properties. The combined effect of the modifications is approximately equivalent to the introduction of one additional double bond19,24,25 (Extended Data Fig. 1). Briefly, bifunctional lipids were loaded into the outer leaflet of the plasma membrane and fixed via subsequent ultraviolet (UV) light and chemical cross-linking (Fig. 1a). To restrict lipid localization to the plasma membrane, lipids were UV cross-linked shortly after lipid loading, and cells were fixed chemically using a protocol adapted from a previous study26 to ensure conservation of CCP ultrastructure. UV cross-linking time was minimized using high-powered light-emitting diodes (LEDs) at 365 nm with a collimation lens to maximize photon flux (Extended Data Fig. 2a–c and Supplementary Data 1). Subsequently, samples were permeabilized to remove non-cross-linked probes and to allow for immunolabelling of...

Lennartz et al. evaluate lipid enrichment in clathrin-coated pits and observe that the differential lipid partitioning into clathrin-coated pits is largely driven by the lipid asymmetry of the plasma ...

Lennartz et al. evaluate lipid enrichment in clathrin-coated pits and observe that the differential lipid partitioning into clathrin-coated pits is largely driven by the lipid asymmetry of the plasma ...

Communications Biology - Clathrin adaptors drive phase separation in endocytosis and trafficking

The clathrin triskelion is a three-legged structure composed of three clathrin heavy chains (CHC) and three CLCs. It self-assembles into a polyhedral lattice, forming a scaffold on the inner surface of the cell membrane: the clathrin coat22. The N-terminal domain of CHC (NTD-CHC) is responsible for interacting with proteins that contain a Clathrin Binding Motif (CBM)15. CHC lacks significant IDRs and, accordingly, is not predicted to form condensates spontaneously. In contrast, CLC contains an unstructured region known to interact with Sla216 and a long ɑ-helix that binds to the distal domain of CHC. Unlike CHC, CLC is predicted to spontaneously undergo phase separation (see Table 1). To test the hypothesis that clathrin adaptors are prone to induce LLPS, we performed a combined analysis using AI-based modeling for IDR prediction and in vitro phase separation experiments of adaptors known to bind clathrin and participate in endocytosis or trans-Golgi network (TGN) trafficking. Our screening identified several proteins with the potential to undergo phase separation, based on predictions from FuzDrop23,24, with a probability of spontaneous LLPS greater than 0.6 (see Table 1, and Supplementary Table S1). Supplementary Table S1 includes information about the IDRs, specifically highlighting the “Droplet-promoting regions”, those predicted residues to...

The B cell receptor (BCR) drives the differentiation of naive B cells into activated plasma cells that produce antibodies. The BCR consists of a plasma membrane-bound surface immunoglobulin (sIg) that...

The sequential assembly of over 50 proteins during clathrin-mediated endocytosis (CME) is well documented in yeast. However, the mechanisms governing this ordered recruitment remain poorly understood....

The sequential assembly of over 50 proteins during clathrin-mediated endocytosis (CME) is well documented in yeast. However, the mechanisms governing this ordered recruitment remain poorly understood....