ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience.

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brain.

Molecular and mesoscopic geometries in autophagosome generation. A review.

Autophagy is an essential process in cell self-repair and survival. The centre of the autophagic event is the generation of the so-called autophagosome (AP), a vesicle surrounded by a double membrane (two bilayers). The AP delivers its cargo to a lysosome, for degradation and re-use of the hydrolysis products as new building blocks. AP formation is a very complex event, requiring dozens of specific proteins, and involving numerous instances of membrane biogenesis and architecture, including membrane fusion and fission. Many stages of AP generation can be rationalised in terms of curvature, both the molecular geometry of lipids interpreted in terms of 'intrinsic curvature', and the overall mesoscopic curvature of the whole membrane, as observed with microscopy techniques. The present contribution intends to bring together the worlds of biophysics and cell biology of autophagy, in the hope that the resulting cross-pollination will generate abundant fruit.

Molecular mechanism for kinesin-1 direct membrane recognition.

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1-dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1-mediated organelle transport.

Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors.

Current methodologies for targeting the mitochondrial genome for research and/or therapy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. A molecular toolbox for CRISPR-mediated mitochondrial genome editing is desirable, as this could enable targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes. Such 'MitoCRISPR' systems described to date lack reproducibility and independent corroboration. We have explored the requirements for MitoCRISPR in human cells by CRISPR nuclease engineering, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of guide (g)RNA modifications for mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and effects on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Our data link mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary for MitoCRISPR.

A heterodimeric SNX4--SNX7 SNX-BAR autophagy complex coordinates ATG9A trafficking for efficient autophagosome assembly.

The sorting nexins (SNXs) are a family of peripheral membrane proteins that direct protein trafficking decisions within the endocytic network. Emerging evidence in yeast and mammalian cells implicates a subgroup of SNXs in selective and non-selective forms of autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that the SNX-BAR protein SNX4 is needed for efficient LC3 (also known as MAP1LC3) lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and hetero-dimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30 that associate with tubulovesicular endocytic membranes. Detailed image-based analysis during the early stages of autophagosome assembly reveals that SNX4-SNX7 is an autophagy-specific SNX-BAR heterodimer, required for efficient recruitment and/or retention of core autophagy regulators at the nascent isolation membrane. SNX4 partially colocalises with juxtanuclear ATG9A-positive membranes, with our data linking the autophagy defect upon SNX4 disruption to the mis-trafficking and/or retention of ATG9A in the Golgi region. Taken together, our findings show that the SNX4-SNX7 heterodimer coordinates ATG9A trafficking within the endocytic network to establish productive autophagosome assembly sites, thus extending knowledge of SNXs as positive regulators of autophagy.

Biophysical Studies of LC3 Family Proteins.

Autophagy is an important cellular process in which cell components are degraded in a controlled way and their building blocks are recycled into new macromolecules. Autophagy starts within a double-membrane container, the autophagosome, itself the result of a number of interconversions of cell membranous elements. In our recent work, we have described reconstituted model systems for the interactions of autophagy proteins with membrane lipid bilayers and for the autophagy protein-mediated vesicle tethering and fusion, with the aim of ultimately reconstituting the autophagosome formation. The present chapter describes in detail (a) the steps required for the preparation of semisynthetic lipid vesicles (liposomes), including giant unilamellar vesicles, (b) ultracentrifugation and fluorescence methods for assaying protein binding to membranes, and (c) procedures for assessing vesicle-vesicle aggregation and fusion. The latter include methods for intervesicular total lipid mixing, mixing of lipids in the vesicle inner monolayers, and aqueous contents mixing.

Human ATG3 binding to lipid bilayers: role of lipid geometry, and electric charge.

Specific protein-lipid interactions lead to a gradual recruitment of AuTophaGy-related (ATG) proteins to the nascent membrane during autophagosome (AP) formation. ATG3, a key protein in the movement of LC3 towards the isolation membrane, has been proposed to facilitate LC3/GABARAP lipidation in highly curved membranes. In this work we have performed a biophysical study of human ATG3 interaction with membranes containing phosphatidylethanolamine, phosphatidylcholine and anionic phospholipids. We have found that ATG3 interacts more strongly with negatively-charged phospholipid vesicles or nanotubes than with electrically neutral model membranes, cone-shaped anionic phospholipids (cardiolipin and phosphatidic acid) being particularly active in promoting binding. Moreover, an increase in membrane curvature facilitates ATG3 recruitment to membranes although addition of anionic lipid molecules makes the curvature factor relatively less important. The predicted N-terminus amphipathic α-helix of ATG3 would be responsible for membrane curvature detection, the positive residues Lys 9 and 11 being essential in the recognition of phospholipid negative moieties. We have also observed membrane aggregation induced by ATG3 in vitro, which could point to a more complex function of this protein in AP biogenesis. Moreover, in vitro GABARAP lipidation assays suggest that ATG3-membrane interaction could facilitate the lipidation of ATG8 homologues.

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B, GABARAPL2 and GABARAP.

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization.

Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ9-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.

Lipid Geometry and Bilayer Curvature Modulate LC3/GABARAP-Mediated Model Autophagosomal Elongation.

Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane-bound structures called autophagosomes (AP), which engulf material to be degraded in lytic compartments. How APs form, especially how the membrane expands and eventually closes upon itself, is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Here, we used two minimal reconstituted systems (enzymatic and chemical conjugation) to compare the ability of human ATG8 homologs (LC3, GABARAP, and GATE-16) to mediate membrane fusion. We found that both enzymatically and chemically lipidated forms of GATE-16 and GABARAP proteins promote extensive membrane tethering and fusion, whereas lipidated LC3 does so to a much lesser extent. Moreover, we characterize the GATE-16/GABARAP-mediated membrane fusion as a phenomenon of full membrane fusion, independently demonstrating vesicle aggregation, intervesicular lipid mixing, and intervesicular mixing of aqueous content, in the absence of vesicular content leakage. Multiple fusion events give rise to large vesicles, as seen by cryo-electron microscopy observations. We also show that both vesicle diameter and selected curvature-inducing lipids (cardiolipin, diacylglycerol, and lyso-phosphatidylcholine) can modulate the fusion process, smaller vesicle diameters and negative intrinsic curvature lipids (cardiolipin, diacylglycerol) facilitating fusion. These results strongly support the hypothesis of a highly bent structural fusion intermediate (stalk) during AP biogenesis and add to the growing body of evidence that identifies lipids as important regulators of autophagy.