A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane.

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (nPR), a ligand-activated transcription factor. A small fraction of progesterone receptor is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα, mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of nPR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the nPR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular progesterone receptor (iPR). This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here, we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent from hormone binding to iPR. We found that a membrane-attached progestin can activate mbPR, the ERK signaling pathway leading to iPR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

Tensing Flipper: Photosensitized Manipulation of Membrane Tension, Lipid Phase Separation, and Raft Protein Sorting in Biological Membranes.

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

Hexagonal Plasmonic Arrays for High-Throughput Multicolor Single-Molecule Studies.

Nanophotonic biosensors offer exceptional sensitivity in the presence of strong background signals by enhancing and confining light in subwavelength volumes. In the field of nanophotonic biosensors, antenna-in-box (AiB) designs consisting of a nanoantenna within a nanoaperture have demonstrated remarkable single-molecule fluorescence detection sensitivities under physiologically relevant conditions. However, their full potential has not yet been exploited as current designs prohibit insightful correlative multicolor single-molecule studies and are limited in terms of throughput. Here, we overcome these constraints by introducing aluminum-based hexagonal close-packed AiB (HCP-AiB) arrays. Our approach enables the parallel readout of over 1000 HCP-AiBs with multicolor single-molecule sensitivity up to micromolar concentrations using an alternating three-color excitation scheme and epi-fluorescence detection. Notably, the high-density HCP-AiB arrays not only enable high-throughput studies at micromolar concentrations but also offer high single-molecule detection probabilities in the nanomolar range. We demonstrate that robust and alignment-free correlative multicolor studies are possible using optical fiducial markers even when imaging in the low millisecond range. These advancements pave the way for the use of HCP-AiB arrays as biosensor architectures for high-throughput multicolor studies on single-molecule dynamics.

Sorting of secretory proteins at the trans-Golgi network by human TGN46.

Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.