The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

Triglyceride-rich lipoproteins (TRLs) undergo lipolysis by lipoprotein lipase (LPL), an enzyme that is transported to the capillary lumen by an endothelial cell protein, GPIHBP1. For LPL-mediated lipolysis to occur, TRLs must bind to the lumen of capillaries. This process is often assumed to involve heparan sulfate proteoglycans (HSPGs), but we suspected that TRL margination might instead require GPIHBP1. Indeed, TRLs marginate along the heart capillaries of wild-type but not Gpihbp1−/− mice, as judged by fluorescence microscopy, quantitative assays with infrared-dye–labeled lipoproteins, and EM tomography. Both cell culture and in vivo studies showed that TRL margination depends on LPL bound to GPIHBP1. Of note, the expression of LPL by endothelial cells in Gpihbp1−/− mice did not restore defective TRL margination, implying that the binding of LPL to HSPGs is ineffective in promoting TRL margination. Our studies show that GPIHBP1-bound LPL is the main determinant of TRL margination.

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.innate host defense | de novo protein design | nanometrology | antibiotics | nanoscopy

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, remains the world's deadliest infectious disease. Sterilising chemotherapy requires at least six months of multidrug therapy. Difficulty visualising the subcellular localisation of antibiotics in infected host cells means that it is unclear whether antibiotics penetrate into all mycobacteria-containing compartments in the cell. Here, we combine correlated light, electron and ion microscopy to image the distribution of Bedaquiline in infected human macrophages at sub-micrometre resolution. Bedaquiline accumulated primarily in host cell lipid droplets, but heterogeneously in mycobacteria within a variety of intracellular compartments. Furthermore, lipid droplets did not sequester antibiotic but constituted a transferable reservoir that enhanced antibacterial efficacy. Thus, strong lipid binding facilitated drug trafficking by host organelles to an intracellular target during antimicrobial treatment.Mycobacterium tuberculosis (Mtb) can persist in multiple intracellular niches within human * Correspondence to: max.

No abstract