Macroautophagy/autophagy is a cellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane organelles called autophagosomes. Lipidation of ubiquitin-like LC3/GABARAP proteins on the autophagosome membrane is important for autophagy. The cysteine protease ATG4 executes 2 LC3/GABARAP processing events: priming of newly synthesized pro-LC3/GABARAP to enable subsequent lipidation, and delipidation/deconjugation of lipidated LC3/GABARAP (the exact purpose of which is unclear in mammals). Four ATG4 isoforms (ATG4A to ATG4D) exist in mammals; however, the functional redundancy of these proteins in cells is poorly understood. Here we show that human HAP1 and HeLa cells lacking ATG4B exhibit a severe but incomplete defect in LC3/GABARAP processing and autophagy. By further genetic depletion of ATG4 isoforms using CRISPR-Cas9 and siRNA we uncover that ATG4A, ATG4C and ATGD all contribute to residual priming activity, which is sufficient to enable lipidation of endogenous GABARAPL1 on autophagic structures. We also demonstrate that expressing high levels of pre-primed LC3B in ATG4-deficient cells can rescue a defect in autophagic degradation of the cargo receptor SQSTM1/p62, suggesting that delipidation by human ATG4 is not essential for autophagosome formation and fusion with lysosomes. Overall, our study provides a comprehensive characterization of ATG4 isoform function during autophagy in human cells. Abbreviations: Atg: autophagy-related; baf A1: bafilomycin A_1; CASP3: caspase 3; CLEM: correlative light and electron microscopy; CMV: cytomegalovirus; CRISPR: clustered regularly interspaced short palindromic repeats; DKO: double knockout; EGFP: enhanced green fluorescent protein; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GFP: green fluorescent protein; HB: homogenization buffer; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3 interacting region; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; N.A.: numerical aperture; NEM: N-ethylmaleimide; PDHA1: pyruvate dehydrogenase E1 alpha 1 subunit; PLD: phospholipase D; PE: phosphatidylethanolamine; RLUC: Renilla luciferase; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TKO: triple knockout; ULK1: unc-51 like autophagy activating kinase 1; VCL: vinculin; WT: wild-type

Upon induction of autophagy, the ubiquitin-like protein LC3 is conjugated to phosphatidylethanolamine (PE) on the inner and outer membrane of autophagosomes to allow cargo selection and autophagosome formation. LC3 undergoes two processing steps, the proteolytic cleavage of pro-LC3 and the de-lipidation of LC3-PE from autophagosomes, both executed by the same cysteine protease ATG4. How ATG4 activity is regulated to co-ordinate these events is currently unknown. Here we find that ULK1, a protein kinase activated at the autophagosome formation site, phosphorylates human ATG4B on serine 316. Phosphorylation at this residue results in inhibition of its catalytic activity in vitro and in vivo. On the other hand, phosphatase PP2A-PP2R3B can remove this inhibitory phosphorylation. We propose that the opposing activities of ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation provide a phospho-switch that regulates the cellular activity of ATG4B to control LC3 processing.

Llamas (Lama glama) naturally produce heavy chain–only antibodies (Abs) in addition to conventional Abs. The variable regions (VHH) in these heavy chain–only Abs demonstrate comparable affinity and specificity for antigens to conventional immunoglobulins despite their much smaller size. To date, immunizations in humans and animal models have yielded only Abs with limited ability to neutralize HIV-1. In this study, a VHH phagemid library generated from a llama that was multiply immunized with recombinant trimeric HIV-1 envelope proteins (Envs) was screened directly for HIV-1 neutralization. One VHH, L8CJ3 (J3), neutralized 96 of 100 tested HIV-1 strains, encompassing subtypes A, B, C, D, BC, AE, AG, AC, ACD, CD, and G. J3 also potently neutralized chimeric simian-HIV strains with HIV subtypes B and C Env. The sequence of J3 is highly divergent from previous anti–HIV-1 VHH and its own germline sequence. J3 achieves broad and potent neutralization of HIV-1 via interaction with the CD4-binding site of HIV-1 Env. This study may represent a new benchmark for immunogens to be included in B cell–based vaccines and supports the development of VHH as anti–HIV-1 microbicides.

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 μm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures ("quanta") of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities.