Endocytosis mediates the cellular uptake of micronutrients and the turnover of plasma membrane proteins. Clathrin-mediated endocytosis is the major uptake pathway in resting cells, but several clathrin-independent endocytic routes exist in parallel. One such pathway, fast endophilin-mediated endocytosis (FEME), is not constitutive but triggered upon activation of certain receptors, including the β adrenergic receptor. FEME activates promptly following stimulation as endophilin is pre-enriched by the phosphatidylinositol-3,4-bisphosphate-binding protein lamellipodin. However, in the absence of stimulation, endophilin foci abort and disassemble after a few seconds. Looking for additional proteins involved in FEME, we found that 20 out of 65 BAR domain-containing proteins tested colocalized with endophilin spots. Among them, FBP17 and CIP4 prime the membrane of resting cells for FEME by recruiting the 5'-lipid phosphatase SHIP2 and lamellipodin to mediate the local production of phosphatidylinositol-3,4-bisphosphate and endophilin pre-enrichment. Membrane-bound GTP-loaded Cdc42 recruits FBP17 and CIP4, before being locally deactivated by RICH1 and SH3BP1 GTPase-activating proteins. This generates the transient assembly and disassembly of endophilin spots, which lasts 5-10 seconds. This mechanism periodically primes patches of the membrane for prompt responses upon FEME activation.

In addition to the classical pathway of secretion, some transmembrane proteins reach the plasma membrane through alternative routes. Several proteins transit through endosomes and are exported in a Rab8-, Rab10-, and/or Rab11-dependent manner. GRAFs are membrane-binding proteins associated with tubules and vesicles. We found extensive colocalization of GRAF1b/2 with Rab8a/b and partial with Rab10. We identified MICAL1 and WDR44 as direct GRAF-binding partners. MICAL1 links GRAF1b/2 to Rab8a/b and Rab10, and WDR44 binds Rab11. Endogenous WDR44 labels a subset of tubular endosomes, which are closely aligned with the ER via binding to VAPA/B. With its BAR domain, GRAF2 can tubulate membranes, and in its absence WDR44 tubules are not observed. We show that GRAF2 and WDR44 are essential for the export of neosynthesized E-cadherin, MMP14, and CFTR ΔF508, three proteins whose exocytosis is sensitive to ER stress. Overexpression of dominant negative mutants of GRAF1/2, WDR44, and MICAL1 also interferes with it, facilitating future studies of Rab8/10/11–dependent exocytic pathways of central importance in biology.

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.

In the version of this Letter originally published, the name of co-author Safa Lucken-Ardjomande Häsler was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

Apoptosis is a form of programmed cell death required for the development and for the proper functioning of multicellular organisms. It is defined by a combination of morphological and biochemical modifications that result from the activation of a family of proteases called caspases. Several pathways can lead to caspase activation and they often involve the release of apoptogenic factors normally sequestered in the mitochondrial intermembrane space. Complete release of mitochondrial pro-apoptotic factors ultimately results in cell death, whether in a caspase-dependent or independent manner. A tight control of mitochondrial permeability is therefore essential. Mutations of regulators of the process, such as proteins of the Bcl-2 family, have indeed been reported in many cancers. In addition, the contributions of lipids, both as regulators of protein activities and as components of the pore itself, are starting to be unravelled. Early on, the role of the mitochondria-specific phospholipid cardiolipin as a targeting signal for pro-apoptotic proteins of the Bcl-2 family was discovered. This role was then expanded since it was shown that cardiolipin also supports conformational changes undergone by proteins of the Bcl-2 family, serves as a docking station for additional pro-apoptotic factors, and is essential for the permeabilisation of synthetic liposomes by activated Bax and Bak. More recently, cholesterol, whose level is increased in most cancer cells, was shown to contribute to their resistance to cytotoxic stresses. Reducing cholesterol levels might therefore represent an interesting novel target to sensitize cancer cells to chemotherapeutic agents.