In eukaryotic cells, various membrane-bound organelles compartmentalize diverse cellular activities in a spatially and temporally controlled manner. Numerous membraneless organelles assembled via ...liquid-liquid phase separation (LLPS), known as condensates, also facilitate compartmentalization of cellular functions. Emerging evidence shows that these two organelle types interact in many biological processes. Membranes modulate the biogenesis and dynamics of phase-separated condensates by serving as assembly platforms or by forming direct contacts. Phase separation of membrane-associated proteins participates in various trafficking events, such as clustering of vesicles for temporally controlled fusion and storage, and transport of membraneless condensates on membrane-bound organelles. Phase separation also acts in cargo trafficking pathways by sorting and docking cargos for translocon-mediated transport across membranes, by shuttling cargos through the nuclear pore complex, and by triggering the formation of surrounding autophagosomes for delivery to lysosomes. The coordinated actions of membrane-bound and membraneless organelles ensure spatiotemporal control of various cellular functions.
Zhao and Zhang summarize the coordinated interactions between membrane-bound organelles and membraneless condensates in various biological processes. Membranes provide surfaces for assembling condensates and also modulate their dynamics and transport, while protein phase separation regulates the storage and trafficking of membrane-bound organelles and also facilitates protein translocation across membranes.
Macroautophagy involves the sequestration of cytoplasmic contents in a double-membrane autophagosome and their delivery to lysosomes for degradation. In multicellular organisms, nascent ...autophagosomes fuse with vesicles originating from endolysosomal compartments before forming degradative autolysosomes, a process known as autophagosome maturation. ATG8 family members, tethering factors, Rab GTPases, and SNARE proteins act coordinately to mediate fusion of autophagosomes with endolysosomal vesicles. The machinery mediating autophagosome maturation is under spatiotemporal control and provides regulatory nodes to integrate nutrient availability with autophagy activity. Dysfunction of autophagosome maturation is associated with various human diseases, including neurodegenerative diseases, Vici syndrome, cancer, and lysosomal storage disorders. Understanding the molecular mechanisms underlying autophagosome maturation will provide new insights into the pathogenesis and treatment of these diseases.
Autophagy is a versatile degradation system for maintaining cellular homeostasis whereby cytosolic materials are sequestered in a double-membrane autophagosome and subsequently delivered to ...lysosomes, where they are broken down. In multicellular organisms, newly formed autophagosomes undergo a process called 'maturation', in which they fuse with vesicles originating from endolysosomal compartments, including early/late endosomes and lysosomes, to form amphisomes, which eventually become degradative autolysosomes. This fusion process requires the concerted actions of multiple regulators of membrane dynamics, including SNAREs, tethering proteins and RAB GTPases, and also transport of autophagosomes and late endosomes/lysosomes towards each other. Multiple mechanisms modulate autophagosome maturation, including post-translational modification of key components, spatial distribution of phosphoinositide lipid species on membranes, RAB protein dynamics, and biogenesis and function of lysosomes. Nutrient status and various stresses integrate into the autophagosome maturation machinery to coordinate the progression of autophagic flux. Impaired autophagosome maturation is linked to the pathogenesis of various human diseases, including neurodegenerative disorders, cancer and myopathies. Furthermore, invading pathogens exploit various strategies to block autophagosome maturation, thus evading destruction and even subverting autophagic vacuoles (autophagosomes, amphisomes and autolysosomes) for survival, growth and/or release. Here, we discuss the recent progress in our understanding of the machinery and regulation of autophagosome maturation, the relevance of these mechanisms to human pathophysiology and how they are harnessed by pathogens for their benefit. We also provide perspectives on targeting autophagosome maturation therapeutically.
Autophagy, a self-eating process conserved from yeast to mammals, is critical for maintaining cell homeostasis. It involves the formation of a double-membrane structure, called the autophagosome, and ...its subsequent delivery to lysosomes for degradation of sequestrated materials. Our knowledge about autophagy has greatly expanded over the last two decades, mainly due to studies of a set of autophagy-related (ATG) genes identified from yeast genetic screens. Autophagy in higher eukaryotes is far more complicated, because it involves steps that are not present in yeast. These include the formation of extensive contacts between the ER and the isolation membrane (IM, autophagosome precursor), and the maturation of nascent autophagosomes into degradative autolysosomes via fusion with vesicles generated from the endolysosomal compartment. Recent studies have discovered factors that act at these unique steps, greatly advancing our molecular understanding of autophagy in higher eukaryotes.
Core autophagy genes and human diseases Zhao, Yan G; Zhang, Hong
Current opinion in cell biology,
December 2019, 2019-12-00, 20191201, Volume:
61
Journal Article
Peer reviewed
Autophagy involves the formation of double-membrane autophagosomes and their delivery to lysosomes for degradation. In response to various endogenous and exogenous stimuli, autophagy recycles ...cellular constituents and removes cytotoxic threats such as protein aggregates and damaged organelles to maintain cellular homeostasis. Dysfunctional autophagy has been linked with multiple human diseases, including neurodegenerative diseases, tumorigenesis, diabetes, and immune diseases. Here we focus on human genetic disorders caused by hypomorphic or regulatory mutations in early acting autophagy genes or by mutations in genes acting at autophagosome maturation. Protein aggregates assembled via liquid–liquid phase separation (LLPS) exhibit distinct biophysical properties that are modulated by disease-related mutations. Abnormal phase transition of protein aggregates affects their removal and is associated with the pathogenesis of various neurodegenerative diseases.
The endoplasmic reticulum (ER) is the site of biogenesis of the isolation membrane (IM, autophagosome precursor) and forms extensive contacts with IMs during their expansion into double-membrane ...autophagosomes. Little is known about the molecular mechanism underlying the formation and/or maintenance of the ER/IM contact. The integral ER proteins VAPA and VAPB (VAPs) participate in establishing ER contacts with multiple membranes by interacting with different tethers. Here, we demonstrate that VAPs also modulate ER/IM contact formation. Depletion of VAPs impairs progression of IMs into autophagosomes. Upon autophagy induction, VAPs are recruited to autophagosome formation sites on the ER, a process mediated by their interactions with FIP200 and PI(3)P. VAPs directly interact with FIP200 and ULK1 through their conserved FFAT motifs and stabilize the ULK1/FIP200 complex at the autophagosome formation sites on the ER. The formation of ULK1 puncta is significantly reduced by VAPA/B depletion. VAPs also interact with WIPI2 and enhance the formation of the WIPI2/FIP200 ER/IM tethering complex. Depletion of VMP1, which increases the ER/IM contact, greatly elevates the interaction of VAPs with these autophagy proteins. The VAPB P56S mutation, which is associated with amyotrophic lateral sclerosis, reduces the ULK1/FIP200 interaction and impairs autophagy at an early step, similar to the effect seen in VAPA/B-depleted cells. Our study reveals that VAPs directly interact with multiple ATG proteins, thereby contributing to ER/IM contact formation for autophagosome biogenesis.
Display omitted
•The ER contact proteins VAPA/B directly interact with ULK1/FIP200 via FFAT motifs•VAPs recruit and/or stabilize the association of the ULK1/FIP200 complex on the ER•VAPs interact with WIPI2 to mediate the ER/IM contact•The ALS-associated VAPB P56S mutation impairs autophagy
Zhao et al. demonstrate that the ER-localized proteins VAPA and VAPB participate in ER/isolation membrane contact formation for autophagosome biogenesis. VAPA/B interact with FIP200 and ULK1 through their FFAT motifs to facilitate assembly of the autophagy initiation complex on the ER.
Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or ...deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.
During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific ...autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarcoendoplasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.
Display omitted
•The ER-localized autophagy protein VMP1 modulates ER contact maintenance/disassembly•WIPI2 interacts with the ULK1/FIP200 complex and PI(3)P for ER-IM contact formation•VMP1 controls membrane contact disassembly by promoting SERCA activity•VMP1 prevents formation of the SERCA/PLN/SLN inhibitory complex
Zhao et al. demonstrate that the ER-localized metazoan-specific autophagy protein VMP1 controls ER contacts with IMs and other organelles. VMP1 controls contact maintenance by modulating SERCA activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex.
Mutations in WDR45 and WDR45B cause the human neurological diseases β-propeller protein-associated neurodegeneration (BPAN) and intellectual disability (ID), respectively. WDR45 and WDR45B, along ...with WIPI1 and WIPI2, belong to a WD40 repeat-containing phosphatidylinositol-3-phosphate (PI(3)P)-binding protein family. Their yeast homolog Atg18 forms a complex with Atg2 and is required for autophagosome formation in part by tethering isolation membranes (IMs) (autophagosome precursor) to the endoplasmic reticulum (ER) to supply lipid for IM expansion in the autophagy pathway. The exact functions of WDR45/45B are unclear. We show here that WDR45/45B are specifically required for neural autophagy. In Wdr45/45b-depleted cells, the size of autophagosomes is decreased, and this is rescued by overexpression of ATG2A, providing in vivo evidence for the lipid transfer activity of ATG2-WIPI complexes. WDR45/45B are dispensable for the closure of autophagosomes but essential for the progression of autophagosomes into autolysosomes. WDR45/45B interact with the tether protein EPG5 and target it to late endosomes/lysosomes to promote autophagosome maturation. In the absence of Wdr45/45b, formation of the fusion machinery, consisting of SNARE proteins and EPG5, is dampened. BPAN- and ID-related mutations of WDR45/45B fail to rescue the autophagy defects in Wdr45/45b-deficient cells, possibly due to their impaired binding to EPG5. Promoting autophagosome maturation by inhibiting O-GlcNAcylation increases SNARE complex formation and facilitates the fusion of autophagosomes with late endosomes/lysosomes in Wdr45/45b double knockout (DKO) cells. Thus, our results uncover a novel function of WDR45/45B in autophagosome-lysosome fusion and provide molecular insights into the development of WDR45/WDR45B mutation-associated diseases.
•β-propeller proteins WDR45/45B regulate autophagosome maturation in neural cells•WDR45/45B bind to the tether protein EPG5 and assist its late endosome localization•Disease-related mutations of WDR45/45B impair their function in autophagy•Inhibition of O-GlcNAcylation suppresses the defects in Wdr45/45b-deficient cells
Ji et al. demonstrate that the β-propeller proteins WDR45 and WDR45B modulate autophagosome-lysosome fusion in neural cells. WDR45/45B interact with the tether protein EPG5 for its targeting to late endosomes/lysosomes and therefore promote the formation of SNARE-tether fusion complexes during autophagosome maturation into autolysosomes.
The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes ...targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.
PDF
