Migfilin promotes autophagic flux through direct interaction with SNAP29 and Vamp8.

Autophagy plays a crucial role in cancer cell survival by facilitating the elimination of detrimental cellular components and the recycling of nutrients. Understanding the molecular regulation of autophagy is critical for developing interventional approaches for cancer therapy. In this study, we report that migfilin, a focal adhesion protein, plays a novel role in promoting autophagy by increasing autophagosome-lysosome fusion. We found that migfilin is associated with SNAP29 and Vamp8, thereby facilitating Stx17-SNAP29-Vamp8 SNARE complex assembly. Depletion of migfilin disrupted the formation of the SNAP29-mediated SNARE complex, which consequently blocked the autophagosome-lysosome fusion, ultimately suppressing cancer cell growth. Restoration of the SNARE complex formation rescued migfilin-deficiency-induced autophagic flux defects. Finally, we found depletion of migfilin inhibited cancer cell proliferation. SNARE complex reassembly successfully reversed migfilin-deficiency-induced inhibition of cancer cell growth. Taken together, our study uncovers a new function of migfilin as an autophagy-regulatory protein and suggests that targeting the migfilin-SNARE assembly could provide a promising therapeutic approach to alleviate cancer progression.

LLPS of FXR proteins drives replication organelle clustering for ß-coronaviral proliferation.

ß-Coronaviruses remodel host endomembranes to form double-membrane vesicles (DMVs) as replication organelles (ROs) that provide a shielded microenvironment for viral RNA synthesis in infected cells. DMVs are clustered, but the molecular underpinnings and pathophysiological functions remain unknown. Here, we reveal that host fragile X-related (FXR) family proteins (FXR1/FXR2/FMR1) are required for DMV clustering induced by expression of viral non-structural proteins (Nsps) Nsp3 and Nsp4. Depleting FXRs results in DMV dispersion in the cytoplasm. FXR1/2 and FMR1 are recruited to DMV sites via specific interaction with Nsp3. FXRs form condensates driven by liquid-liquid phase separation, which is required for DMV clustering. FXR1 liquid droplets concentrate Nsp3 and Nsp3-decorated liposomes in vitro. FXR droplets facilitate recruitment of translation machinery for efficient translation surrounding DMVs. In cells depleted of FXRs, SARS-CoV-2 replication is significantly attenuated. Thus, SARS-CoV-2 exploits host FXR proteins to cluster viral DMVs via phase separation for efficient viral replication.

Stay in touch with the endoplasmic reticulum.

The endoplasmic reticulum (ER), which is composed of a continuous network of tubules and sheets, forms the most widely distributed membrane system in eukaryotic cells. As a result, it engages a variety of organelles by establishing membrane contact sites (MCSs). These contacts regulate organelle positioning and remodeling, including fusion and fission, facilitate precise lipid exchange, and couple vital signaling events. Here, we systematically review recent advances and converging themes on ER-involved organellar contact. The molecular basis, cellular influence, and potential physiological functions for ER/nuclear envelope contacts with mitochondria, Golgi, endosomes, lysosomes, lipid droplets, autophagosomes, and plasma membrane are summarized.

DMV biogenesis during ß-coronavirus infection requires autophagy proteins VMP1 and TMEM41B.

Upon entering host cells, ß-coronaviruses specifically induce generation of replication organelles (ROs) from the endoplasmic reticulum (ER) through their nonstructural protein 3 (nsp3) and nsp4 for viral genome transcription and replication. The most predominant ROs are double-membrane vesicles (DMVs). The ER-resident proteins VMP1 and TMEM41B, which form a complex to regulate autophagosome and lipid droplet (LD) formation, were recently shown to be essential for ß-coronavirus infection. Here we report that VMP1 and TMEM41B contribute to DMV generation but function at different steps. TMEM41B facilitates nsp3-nsp4 interaction and ER zippering, while VMP1 is required for subsequent closing of the paired ER into DMVs. Additionally, inhibition of phosphatidylserine (PS) formation by siPTDSS1 partially reverses the DMV and LD defects in VMP1 KO cells, suggesting that appropriate PS levels also contribute to DMV formation. This work provides clues to the mechanism of how host proteins collaborate with viral proteins for endomembrane reshaping to promote viral infection.

VMP1 and TMEM41B are essential for DMV formation during ß-coronavirus infection.

ß-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3-nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.

Endomembrane remodeling in SARS-CoV-2 infection.

During severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the viral proteins intimately interact with host factors to remodel the endomembrane system at various steps of the viral lifecycle. The entry of SARS-CoV-2 can be mediated by endocytosis-mediated internalization. Virus-containing endosomes then fuse with lysosomes, in which the viral S protein is cleaved to trigger membrane fusion. Double-membrane vesicles generated from the ER serve as platforms for viral replication and transcription. Virions are assembled at the ER-Golgi intermediate compartment and released through the secretory pathway and/or lysosome-mediated exocytosis. In this review, we will focus on how SARS-CoV-2 viral proteins collaborate with host factors to remodel the endomembrane system for viral entry, replication, assembly and egress. We will also describe how viral proteins hijack the host cell surveillance system-the autophagic degradation pathway-to evade destruction and benefit virus production. Finally, potential antiviral therapies targeting the host cell endomembrane system will be discussed.

Machinery, regulation and pathophysiological implications of autophagosome maturation.

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.

The BPAN and intellectual disability disease proteins WDR45 and WDR45B modulate autophagosome-lysosome fusion.

WDR45 and WDR45B are ß-propeller proteins belonging to the WIPI (WD repeat domain, phosphoinositide interacting) family. Mutations in WDR45 and WDR45B are genetically linked with beta-propeller protein-associated neurodegeneration (BPAN) and intellectual disability (ID), respectively. WDR45 and WDR45B are homologs of yeast Atg18. Atg18 forms a complex with Atg2 for autophagosome biogenesis, probably by transferring lipids from the ER to phagophores. We revealed that WDR45 and WDR45B are critical for autophagosome-lysosome fusion in neural cells. WDR45 and WDR45B, but not their disease-related mutants, bind to the tether protein EPG5 and facilitate its targeting to late endosomes/lysosomes. In Wdr45 Wdr45b-deficient cells, the formation of tether-SNARE fusion machinery is compromised. The macroautophagy/autophagy deficiency in wdr45 wdr45b DKO cells is ameliorated by suppression of O-GlcNAcylation, which promotes autophagosome maturation. Thus, our results provide insights into the pathogenesis of WDR45- and WDR45B-related neurological diseases.

Atlastin 2/3 regulate ER targeting of the ULK1 complex to initiate autophagy.

Dynamic targeting of the ULK1 complex to the ER is crucial for initiating autophagosome formation and for subsequent formation of ER-isolation membrane (IM; autophagosomal precursor) contact during IM expansion. Little is known about how the ULK1 complex, which comprises FIP200, ULK1, ATG13, and ATG101 and does not exist as a constitutively coassembled complex, is recruited and stabilized on the ER. Here, we demonstrate that the ER-localized transmembrane proteins Atlastin 2 and 3 (ATL2/3) contribute to recruitment and stabilization of ULK1 and ATG101 at the FIP200-ATG13-specified autophagosome formation sites on the ER. In ATL2/3 KO cells, formation of FIP200 and ATG13 puncta is unaffected, while targeting of ULK1 and ATG101 is severely impaired. Consequently, IM initiation is compromised and slowed. ATL2/3 directly interact with ULK1 and ATG13 and facilitate the ATG13-mediated recruitment/stabilization of ULK1 and ATG101. ATL2/3 also participate in forming ER-IM tethering complexes. Our study provides insights into the dynamic assembly of the ULK1 complex on the ER for autophagosome formation.

Vmp1, Vps13D, and Marf/Mfn2 function in a conserved pathway to regulate mitochondria and ER contact in development and disease.

Mutations in Vps13D cause defects in autophagy, clearance of mitochondria, and human movement disorders. Here, we discover that Vps13D functions in a pathway downstream of Vmp1 and upstream of Marf/Mfn2. Like vps13d, vmp1 mutant cells exhibit defects in autophagy, mitochondrial size, and clearance. Through the relationship between vmp1 and vps13d, we reveal a novel role for Vps13D in the regulation of mitochondria and endoplasmic reticulum (ER) contact. Significantly, the function of Vps13D in mitochondria and ER contact is conserved between fly and human cells, including fibroblasts derived from patients suffering from VPS13D mutation-associated neurological symptoms. vps13d mutants have increased levels of Marf/MFN2, a regulator of mitochondrial fusion. Importantly, loss of marf/MFN2 suppresses vps13d mutant phenotypes, including mitochondria and ER contact. These findings indicate that Vps13d functions at a regulatory point between mitochondria and ER contact, mitochondrial fusion and autophagy, and help to explain how Vps13D contributes to disease.

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice.

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.

ß-propeller proteins WDR45 and WDR45B regulate autophagosome maturation into autolysosomes in neural cells.

Mutations in WDR45 and WDR45B cause the human neurological diseases ß-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.

Inositol Polyphosphate Multikinase Inhibits Liquid-Liquid Phase Separation of TFEB to Negatively Regulate Autophagy Activity.

Liquid-liquid phase separation (LLPS) compartmentalizes transcriptional condensates for gene expression, but little is known about how this process is controlled. Here, we showed that depletion of IPMK, encoding inositol polyphosphate multikinase, promotes autophagy and lysosomal function and biogenesis in a TFEB-dependent manner. Cytoplasmic-nuclear trafficking of TFEB, a well-characterized mechanism by which diverse signaling pathways regulate TFEB activity, is not evidently altered by IPMK depletion. We demonstrated that nuclear TFEB forms distinct puncta that colocalize with the Mediator complex and with mRNAs of target lysosomal genes. TFEB undergoes LLPS in vitro. IPMK directly interacts with and inhibits LLPS of TFEB and also dissolves TFEB condensates. Depletion of IPMK increases the number of nuclear TFEB puncta and the co-localization of TFEB with Mediator and mRNAs of target genes. Our study reveals that nuclear-localized IPMK acts as a chaperone to inhibit LLPS of TFEB to negatively control its transcriptional activity.

Phase Separation in Membrane Biology: The Interplay between Membrane-Bound Organelles and Membraneless Condensates.

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.

Role of Wdr45b in maintaining neural autophagy and cognitive function.

Macroautophagy/autophagy functions as a quality control mechanism by degrading misfolded proteins and damaged organelles and plays an essential role in maintaining neural homeostasis. The phosphoinositide phosphatidylinositol-3-phosphate (PtdIns3P) effector Atg18 is essential for autophagosome formation in yeast. Mammalian cells contain four Atg18 homologs, belonging to two subclasses, WIPI1 (WD repeat domain, phosphoinositide interacting 1), WIPI2 and WDR45B/WIPI3 (WD repeat domain 45B), WDR45/WIPI4. The role of Wdr45b in autophagy and in neural homeostasis, however, remains unknown. Recent human genetic studies have revealed a potential causative role of WDR45B in intellectual disability. Here we demonstrated that mice deficient in Wdr45b exhibit motor deficits and learning and memory defects. Histological analysis reveals that wdr45b knockout (KO) mice exhibit a large number of swollen axons and show cerebellar atrophy. SQSTM1- and ubiquitin-positive aggregates, which are autophagy substrates, accumulate in various brain regions in wdr45b KO mice. Double KO mice, wdr45b and wdr45, die within one day after birth and exhibit more severe autophagy defects than either of the single KO mice, suggesting that these two genes act cooperatively in autophagy. Our studies demonstrated that WDR45B is critical for neural homeostasis in mice. The wdr45b KO mice provide a model to study the pathogenesis of intellectual disability.Abbreviations: ACSF: artificial cerebrospinal fluid; AMC: aminomethylcoumarin; BPAN: beta-propeller protein-associated neurodegeneration; CALB1: calbindin 1; CNS: central nervous system; DCN: deep cerebellar nuclei; fEPSP: field excitatory postsynaptic potential; IC: internal capsule; ID: intellectual disability; ISH: in situ hybridization; KO: knockout; LTP: long-term potentiation; MBP: myelin basic protein; MGP: medial globus pallidus; PtdIns3P: phosphoinositide phosphatidylinositol-3-phosphate; WDR45B: WD repeat domain 45B; WIPI1: WD repeat domain, phosphoinositide interacting 1; WT: wild type.

A critical role of VMP1 in lipoprotein secretion.

Lipoproteins are lipid-protein complexes that are primarily generated and secreted from the intestine, liver, and visceral endoderm and delivered to peripheral tissues. Lipoproteins, which are assembled in the endoplasmic reticulum (ER) membrane, are released into the ER lumen for secretion, but its mechanism remains largely unknown. Here, we show that the release of lipoproteins from the ER membrane requires VMP1, an ER transmembrane protein essential for autophagy and certain types of secretion. Loss of vmp1, but not other autophagy-related genes, in zebrafish causes lipoprotein accumulation in the intestine and liver. Vmp1 deficiency in mice also leads to lipid accumulation in the visceral endoderm and intestine. In VMP1-depleted cells, neutral lipids accumulate within lipid bilayers of the ER membrane, thus affecting lipoprotein secretion. These results suggest that VMP1 is important for the release of lipoproteins from the ER membrane to the ER lumen in addition to its previously known functions.

Core autophagy genes and human diseases.

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 ER-Localized Protein DFCP1 Modulates ER-Lipid Droplet Contact Formation.

Very little is known about the spatiotemporal generation of lipid droplets (LDs) from the endoplasmic reticulum (ER) and the factors that mediate ER-LD contacts for LD growth. Using super-resolution grazing incidence structured illumination microscopy (GI-SIM) live-cell imaging, we reveal that upon LD induction, the ER-localized protein DFCP1 redistributes to nascent puncta on the ER, whose formation depends on triglyceride synthesis. These structures move along the ER and fuse to form expanding LDs. Fusion and expansion of DFCP1-labeled nascent structures is controlled by BSCL2. BSCL2 depletion causes accumulation of nascent DFCP1 structures. DFCP1 overexpression increases LD size and enhances ER-LD contacts, while DFCP1 knockdown has the opposite effect. DFCP1 acts as a Rab18 effector for LD localization and interacts with the Rab18-ZW10 complex to mediate ER-LD contact formation. Our study reveals that fusion of DFCP1-labeled nascent structures contributes to initial LD growth and that the DFCP1-Rab18 complex is involved in tethering the ER-LD contact for LD expansion.

Autophagosome maturation: An epic journey from the ER to lysosomes.

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.

Formation and maturation of autophagosomes in higher eukaryotes: a social network.

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.