Thomas Wollert

The physiological role of lipid asymmetry in intracellular membranes remains poorly understood. Here, we show that sphingomyelin (SM), typically confined to the lumen of the trans-Golgi network (TGN), is exposed on its cytoplasmic surface by the action of the Golgi-associated protein, Golgi-associated gamma-adaptin ear–containing adenosine 5′-diphosphate–ribosylation factor–binding protein 1 (GGA1). This exposure is driven by the GGA1 GAT domain, which induces lipid scrambling in a manner dependent on membrane curvature and cholesterol. SM exposure coincides with the exit of mannose 6-phosphate receptors from the TGN, a process essential for lysosomal enzyme trafficking. Furthermore, SM is transferred to autophagic membranes, where it facilitates autophagosome-lysosome fusion. These findings reveal a previously unrecognized role for lipid remodeling in membrane trafficking and autophagy.

Autophagy is a highly conserved catabolic pathway that maintains cellular homeostasis by promoting the degradation of damaged or superfluous cytoplasmic material. A hallmark of autophagy is the generation of membrane cisternae that sequester autophagic cargo. Expansion of these structures allows cargo to be engulfed in a highly selective and exclusive manner. Cytotoxic stress or starvation induces the formation of autophagosomes that sequester bulk cytoplasm instead of selected cargo. This rather nonselective pathway is essential for maintaining vital cellular functions during adverse conditions and is thus a major stress response pathway. Both selective and nonselective autophagy rely on the same molecular machinery. However, due to the different nature of cargo to be sequestered, the involved molecular mechanisms are fundamentally different. Although intense research over the past decades has advanced our understanding of autophagy, fundamental questions remain to be addressed. This review will focus on molecular principles and open questions regarding the formation of omegasomes and phagophores in nonselective mammalian autophagy.

The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to phagophores by an E3 ligase complex comprising ATG12–ATG5 and ATG16L1. Here we combined two complementary reconstitution approaches to reveal the function of LC3B and its ligase complex during phagophore expansion. We found that LC3B forms together with ATG12–ATG5–ATG16L1 a membrane coat that remodels flat membranes into cups that closely resemble phagophores. Mechanistically, we revealed that cup formation strictly depends on a close collaboration between LC3B and ATG16L1. Moreover, only LC3B, but no other member of the ATG8 protein family, promotes cup formation. ATG16L1 truncates that lacked the C-terminal membrane binding domain catalyzed LC3B lipidation but failed to assemble coats, did not promote cup formation and inhibited the biogenesis of non-selective autophagosomes. Our results thus demonstrate that ATG16L1 and LC3B induce and stabilize the characteristic cup-like shape of phagophores.

Autophagy is a fundamental cellular recycling pathway that captures cytoplasmic material by a phagophore membrane and delivers it to lysosomes for degradation. Nonselective phagophores are generated at specialized ER-domains termed omegasomes. Mechanistically, this process is not well understood. Here, we reconstituted the formation of phagophores from purified components on supported lipid bilayers and by ectopic induction of nonselective autophagy at the plasma membrane. We found that enzymatic conjugation of LC3B to model membranes induces the formation of phagophore-like membrane cups. LC3B functions as membrane anchor, ensuring a sustained interaction of the E3-like ligase complex ATG12¬–ATG5-ATG16L1 with membranes by forming extended membrane coats. ATG16L1 is the major membrane remodeling factor that induces positive membrane curvature, promotes cup formation and stabilizes the rim of membrane cups. Redirecting WIPI2 to the plasma membrane by expressing WIPI2-CAAX induced the formation of omegasome-like membrane cisternae to which LC3B was conjugated. Membrane cups, identical to those observed in vitro, emerged from these cisternae. ATG16L1 variants that did not induce the formation of cups in vitro also failed to promote cup formation in vivo and inhibited the biogenesis of nonselective autophagosomes in cells. We thus propose that ATG16L1 drives the formation of nonselective phagophores by shaping flat donor membranes into membrane cups.

In this very interesting study, the authors investigated how toxic protein aggregates that are the causative agents of Parkinson’s disease are propagated between cells. The hallmark of many neurovegetative diseases including Parkinson’s disease is the accumulation of toxic protein aggregates that consists of prion-like protein fibrils. Protein aggregates that consist of α-synuclein (α-Syn) accumulate in neurons of patients suffering from Parkinson’s disease. The propagation of α-Syn fibrils is closely related to the onset of disease symptoms, but the underlying molecular mechanism was not well characterized. Here, the authors applied a combination of super-resolution imaging and correlative light electron microscopy to study the fate of α-Syn aggregates in neural cells using the neuronal catecholaminergic cell line CAD as a model. Aggregation of α-Syn was induced by incubating cells with exogenous fluorescent-labeled aSyn fibrils. The authors found that exogenous α-Syn fibrils reach lysosomal compartments after internalization where they induce lysosomal permeabilization to seed new fibrils. Furthermore, damaged lysosomes are transported to neighboring cells by tunneling nanotubes, which are thin membrane protrusions that bridge α-Syn fibril containing donor cells and healthy acceptor cells. In summary, the study revealed that lysosomes take a central position in the propagation of α-Syn fibrils by acting as “Trojan horses”.

The ongoing pandemic of the new severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused more than one million deaths, overwhelmed many public health systems, and led to a worldwide economic recession. This has raised an unprecedented need to develop antiviral drugs and vaccines, which requires profound knowledge of the fundamental pathology of the virus, including its entry, replication, and release from host cells. The genome of coronaviruses comprises around 30 kb of positive single-stranded RNA, representing one of the largest RNA genomes of viruses. The 5′ part of the genome encodes a large polyprotein, PP1ab, which gives rise to 16 non-structural proteins (nsp1– nsp16). Two proteases encoded in nsp3 and nsp5 cleave the polyprotein into individual proteins. Most nsps belong to the viral replicase complex that promotes replication of the viral genome and translation of structural proteins by producing subgenomic mRNAs. The replicase complexes are found on double-membrane vesicles (DMVs) that contain viral double-stranded RNA. Expression of a small subset of viral proteins, including nsp3 and nsp4, is sufficient to induce formation of these DMVs in human cells, suggesting that both proteins deform host membranes into such structures. We will discuss the formation of DMVs and provide an overview of other membrane remodeling processes that are induced by coronaviruses.

Despite increased research on coronaviruses, we lack understanding of the molecular mechanisms governing the formation of syncytia in the lungs of patients with severe damage from COVID-19. This paper by Buchrieser et al. shows that, if the spike (S)-protein from severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) is expressed in human cells and transported by the canonical secretory pathway to the plasma membrane, it participates in the formation of multinucleated syncytial pneumocytes by fusing with angiotensin converting enzyme 2 (ACE2)-positive neighboring cells. The authors show that interferon‐induced transmembrane proteins (IFITMs), in particular IFITM1, was able to inhibit S‐mediated fusion, whereas the serine protease TMPRSS2 prompted fusion. The study provides the foundation to investigate the role of syncytia in viral infection, particularly in viral spread and viral immune evasion. This Recommendation is of an article referenced in a Faculty Review also written by Thomas Wollert.

Infection with coronavirus (CoV) induces the production of various membrane structures, but until now it was unclear which of them housed viral RNA synthesis. Now, this work by Snijder et al. identifies double-membrane vesicles (DMVs) as the focal point for this essential process, and thus highlights these structures as being a viable target for novel antiviral drugs to combat infection with severe acute respiratory syndrome-CoV 2 (SARS-CoV-2). The authors analysed various different types of viral replication organelle from several coronaviruses and revealed common features. Viral RNA synthesis was found to be located solely in DMVs using metabolic labeling of newly synthesized viral RNA and subsequent quantitative electron microscopy (EM) autoradiography. The authors suggest that coronavirus double stranded RNA intermediates are shielded within double membrane vesicles in order to evade interferon-1-dependent antiviral immune response. This Recommendation is of an article referenced in a Faculty Review also written by Thomas Wollert.