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In biology, shape and function are related. Therefore, it is important to understand how membrane shape is generated, stabilised and sensed by proteins and how this relates to organelle function. Here, we present an assay that can detect curvature preference and membrane remodelling with free-floating liposomes using protein concentrations in physiologically relevant ranges. The assay reproduced known curvature preferences of BAR domains and allowed the discovery of high-curvature preference for the PH domain of AKT and the FYVE domain of HRS (also known as HGS). In addition, our method reproduced the membrane vesiculation activity of the ENTH domain of epsin-1 (EPN1) and showed similar activity for the ANTH domains of PiCALM and Hip1R. Finally, we found that the curvature sensitivity of the N-BAR domain of endophilin inversely correlates to membrane charge and that deletion of its N-terminal amphipathic helix increased its curvature specificity. Thus, our method is a generally applicable qualitative method for assessing membrane curvature sensing and remodelling by proteins., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Membrane curvature is an important parameter in defining the morphology of cells, organelles and local membrane subdomains. Transport intermediates have simpler shapes, being either spheres or tubules. The generation and maintenance of curvature is of central importance for maintaining trafficking and cellular functions. It is possible that local shapes in complex membranes could help to define local subregions. In this Cell Science at a Glance article and accompanying poster, we summarize how generating, sensing and maintaining high local membrane curvature is an active process that is mediated and controlled by specialized proteins using general mechanisms: (i) changes in lipid composition and asymmetry, (ii) partitioning of shaped transmembrane domains of integral membrane proteins or protein or domain crowding, (iii) reversible insertion of hydrophobic protein motifs, (iv) nanoscopic scaffolding by oligomerized hydrophilic protein domains and, finally, (v) macroscopic scaffolding by the cytoskeleton with forces generated by polymerization and by molecular motors. We also summarize some of the discoveries about the functions of membrane curvature, where in addition to providing cell or organelle shape, local curvature can affect processes like membrane scission and fusion as well as protein concentration and enzyme activation on membranes., (© 2015. Published by The Company of Biologists Ltd.)

Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We found that GRAF1a, the longest GRAF1 isoform (GRAF1 is also known as ARHGAP26), was enriched in the brains of neonates. Endogenous GRAF1a was found on lipid droplets in oleic-acid-fed primary glial cells. Exclusive localization required a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promoted lipid droplet clustering, inhibited droplet mobility and severely perturbed lipolysis following the chase of cells overloaded with fatty acids. Under these conditions, GRAF1a concentrated at the interface between lipid droplets. Although GRAF1-knockout mice did not show any gross abnormal phenotype, the total lipid droplet volume that accumulated in GRAF1(-/-) primary glia upon incubation with fatty acids was reduced compared to GRAF1(+/+) cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis., (© 2014. Published by The Company of Biologists Ltd.)

Sorting nexins are a large family of phox-homology-domain-containing proteins that have been implicated in the control of endosomal sorting. Sorting nexin-1 is a component of the mammalian retromer complex that regulates retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network. In yeast, retromer is composed of Vps5p (the orthologue of sorting nexin-1), Vps17p (a related sorting nexin) and a cargo selective subcomplex composed of Vps26p, Vps29p and Vps35p. With the exception of Vps17p, mammalian orthologues of all yeast retromer components have been identified. For Vps17p, one potential mammalian orthologue is sorting nexin-2. Here we show that, like sorting nexin-1, sorting nexin-2 binds phosphatidylinositol 3-monophosphate and phosphatidylinositol 3,5-bisphosphate, and possesses a Bin/Amphiphysin/Rvs domain that can sense membrane curvature. However, in contrast to sorting nexin-1, sorting nexin-2 could not induce membrane tubulation in vitro or in vivo. Functionally, we show that endogenous sorting nexin-1 and sorting nexin-2 co-localise on high curvature tubular elements of the 3-phosphoinositide-enriched early endosome, and that suppression of sorting nexin-2 does not perturb the degradative sorting of receptors for epidermal growth factor or transferrin, nor the steady-state distribution of the cation-independent mannose 6-phosphate receptor. However, suppression of sorting nexin-2 results in a subtle alteration in the kinetics of cation-independent mannose 6-phosphate receptor retrieval. These data suggest that although sorting nexin-2 may be a component of the retromer complex, its presence is not essential for the regulation of endosome-to-trans Golgi network retrieval of the cation-independent mannose 6-phosphate receptor.