The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension.

Human WIPI ß-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan.

Multiparametric platform for profiling lipid trafficking in human leukocytes.

Systematic insight into cellular dysfunction can improve understanding of disease etiology, risk assessment, and patient stratification. We present a multiparametric high-content imaging platform enabling quantification of low-density lipoprotein (LDL) uptake and lipid storage in cytoplasmic droplets of primary leukocyte subpopulations. We validate this platform with samples from 65 individuals with variable blood LDL-cholesterol (LDL-c) levels, including familial hypercholesterolemia (FH) and non-FH subjects. We integrate lipid storage data into another readout parameter, lipid mobilization, measuring the efficiency with which cells deplete lipid reservoirs. Lipid mobilization correlates positively with LDL uptake and negatively with hypercholesterolemia and age, improving differentiation of individuals with normal and elevated LDL-c. Moreover, combination of cell-based readouts with a polygenic risk score for LDL-c explains hypercholesterolemia better than the genetic risk score alone. This platform provides functional insights into cellular lipid trafficking and has broad possible applications in dissecting the cellular basis of metabolic disorders.

Low-Density Lipoprotein Internalization, Degradation and Receptor Recycling Along Membrane Contact Sites.

Low-density lipoprotein (LDL) internalization, degradation, and receptor recycling is a fundamental process underlying hypercholesterolemia, a high blood cholesterol concentration, affecting more than 40% of the western population. Membrane contact sites influence endosomal dynamics, plasma membrane lipid composition, and cellular cholesterol distribution. However, if we focus on LDL-related trafficking events we mostly discuss them in an isolated fashion, without cellular context. It is our goal to change this perspective and to highlight that all steps from LDL internalization to receptor recycling are likely associated with dynamic membrane contact sites in which endosomes engage with the endoplasmic reticulum and other organelles.

High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants.

The human Niemann-Pick C1 (NPC1) gene encoding a 1278 amino acid protein is very heterogeneous. While some variants represent benign polymorphisms, NPC disease carriers and patients may possess rare variants, whose functional importance remains unknown. An NPC1 cDNA construct known as NPC1 wild-type variant (WT-V), distributed between laboratories and used as a WT control in several studies, also contains changes regarding specific amino acids compared to the NPC1 Genbank reference sequence. To improve the dissection of subtle functional differences, we generated human cells stably expressing NPC1 variants from the AAVS1 safe-harbor locus on an NPC1-null background engineered by CRISPR/Cas9 editing. We then employed high-content imaging with automated image analysis to quantitatively assess LDL-induced, time-dependent changes in lysosomal cholesterol content and lipid droplet formation. Our results indicate that the L472P change present in NPC1 WT-V compromises NPC1 functionality in lysosomal cholesterol export. All-atom molecular dynamics simulations suggest that the L472P change alters the relative position of the NPC1 middle and the C-terminal luminal domains, disrupting the recently characterized cholesterol efflux tunnel. These results reveal functional defects in NPC1 WT-V and highlight the strength of simulations and quantitative imaging upon stable protein expression in elucidating subtle differences in protein function.

Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

Role for formin-like 1-dependent acto-myosin assembly in lipid droplet dynamics and lipid storage.

Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoing homotypic clustering and fusion. In non-adipocytic cells with numerous LDs this is balanced by poorly understood droplet dissociation mechanisms. We identify non-muscle myosin IIa (NMIIa/MYH-9) and formin-like 1 (FMNL1) in the LD proteome. NMIIa and actin filaments concentrate around LDs, and form transient foci between dissociating LDs. NMIIa depletion results in decreased LD dissociations, enlarged LDs, decreased hydrolysis and increased storage of TGs. FMNL1 is required for actin assembly on LDs in vitro and for NMIIa recruitment to LDs in cells. We propose a novel acto-myosin structure regulating lipid storage: FMNL1-dependent assembly of myosin II-functionalized actin filaments on LDs facilitates their dissociation, thereby affecting LD surface-to-volume ratio and enzyme accessibility to TGs. In neutrophilic leucocytes from MYH9-related disease patients NMIIa inclusions are accompanied by increased lipid storage in droplets, suggesting that NMIIa dysfunction may contribute to lipid imbalance in man.

LDL-cholesterol transport to the endoplasmic reticulum: current concepts.

PURPOSE OF REVIEW: In this article, we summarize the present information related to the export of LDL-derived cholesterol from late endosomes, with a focus on Nieman-Pick disease, type C1 (NPC1) cholesterol delivery toward the endoplasmic reticulum (ER). We review data suggesting that several pathways may operate in parallel, including membrane transport routes and membrane contact sites (MCSs). RECENT FINDINGS: There is increasing appreciation that MCSs provide an important mechanism for intermembrane lipid transfer. In late endosome-ER contacts, three protein bridges involving oxysterol binding protein related protein (ORP)1L-vesicle associated membrane protein-associated protein (VAP), steroidogenic acute regulatory protein (StAR)D3-VAP and ORP5-NPC1 proteins have been reported. How much they contribute to the flux of LDL-cholesterol to the ER is currently open. Studies for lipid transfer via MCSs have been most advanced in Saccharomyces cerevisiae. Recently, a new sterol-binding protein family conserved between yeast and man was identified. Its members localize at MCSs and were named lipid transfer protein anchored at membrane contact sites (Lam) proteins. In yeast, sterol transfer between the ER and the yeast lysosome may be facilitated by a Lam protein. SUMMARY: Increasing insights into the role of MCSs in directional sterol delivery between membranes propose that they might provide routes for LDL-cholesterol transfer to the ER. Future work should reveal which specific contacts may operate for this, and how they are controlled by cholesterol homeostatic machineries.

D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage.

We generated a highly deuterated cholesterol analog (D38-cholesterol) and demonstrated its use for selective vibrational imaging of cholesterol storage in mammalian cells. D38-cholesterol produces detectable signals in stimulated Raman scattering (SRS) imaging, is rapidly taken up by cells, and is efficiently metabolized by acyl-CoA cholesterol acyltransferase to form cholesteryl esters. Using hyperspectral SRS imaging of D38-cholesterol, we visualized cholesterol storage in lipid droplets. We found that some lipid droplets accumulated preferentially unesterified D38-cholesterol, whereas others stored D38-cholesteryl esters. In steroidogenic cells, D38-cholesteryl esters and triacylglycerols were partitioned into distinct sets of lipid droplets. Thus, hyperspectral SRS imaging of D38-cholesterol demonstrates a heterogeneous incorporation of neutral lipid species, i.e., free cholesterol, cholesteryl esters, and triacylglycerols, between individual lipid droplets in a cell.

Sterol liganding of OSBP-related proteins (ORPs) regulates the subcellular distribution of ORP-VAPA complexes and their impacts on organelle structure.

Oxysterol-binding protein (OSBP) and its homologues (ORPs) are lipid-binding/transfer proteins with affinity for oxysterols, cholesterol and glycerophospholipids. In addition to a ligand-binding domain, a majority of the ORPs carry a pleckstrin homology domain that targets organelle membranes via phosphoinositides, and a motif targeting the endoplasmic reticulum (ER) via VAMP-associated proteins (VAPs). We employed here Bimolecular Fluorescence Complementation (BiFC) to systematically assess the effects of sterol manipulation of HuH7 cells on complexes of established sterol-binding ORPs with their ER receptor, VAMP-associated protein A (VAPA). Depletion of cellular cholesterol with lipoprotein-deficient medium and Mevastatin caused concentration of OSBP-VAPA complexes and Golgi complex markers at a juxtanuclear position, an effect reversed by low-density lipoprotein treatment. A similar redistribution of OSBP-VAPA but not of sterol-binding deficient mutant OSBP(ΔELSK)-VAPA, occurred upon treatment with the high-affinity ligand, 25-hydroxycholesterol (25OHC), which reduced total and free cholesterol. ORP2-VAPA complexes, which localize in untreated cells at blob-like ER structures with associated lipid droplets, were redistributed upon treatment with the ORP2 ligand 22(R)OHC to a diffuse cytoplasmic/ER pattern and the plasma membrane. Analogously, distribution of ORP4L-VAPA complexes between the plasma membrane and vimentin intermediate filament associated compartments was modified by statin or 25OHC treatment. The treatments resulted in loss of vimentin co-localization, and sterol-binding deficient ORP4L(ΔELSR)-VAPA localized predominantly to the plasma membrane. In conclusion, treatment with statin or oxysterol ligands modify the subcellular targeting of ORP-VAPA complexes, consistent with the notion that this machinery controls lipid homeostasis and signaling at organelle interfaces.

Polarized THG microscopy identifies compositionally different lipid droplets in mammalian cells.

Cells store excess lipids as two major compounds, triacylglycerols (TAGs) and cholesteryl esters (CEs), inside lipid droplets (LDs). The degree of lipid ordering is considered to play a major role in the mobility and enzymatic processing of lipids in LDs. Here, we provide evidence that polarized third-harmonic generation (THG) microscopy distinguishes between native TAG- and CE-enriched LDs in cells due to the different ordering of the two lipid species. We first demonstrate that the responses from synthetic TAG- and CE-enriched LDs using THG microscopy with linear and circular polarizations differ according to their different intrinsic ordering. We then employ simulations to dissect how polarization effects influence the THG from an isotropic LD. Finally, we induce TAG- and CE-enriched LDs in murine macrophages and demonstrate that polarized THG responses increase in a nonlinear fashion with increasing CE/TAG ratio. This suggests that with an increasing CE content, there is a rather sharp transition toward increased LD ordering. Our results demonstrate that polarized THG microscopy enables label-free quantitative analysis of LD ordering and discriminates between compositionally different LDs in intact mammalian cells.

WIPI ß-propellers at the crossroads of autophagosome and lipid droplet dynamics.

Macroautophagy (autophagy hereafter) is an evolutionarily highly conserved catabolic process activated by eukaryotes in order to counteract cellular starvation. Autophagy leads to bulk degradation of cytoplasmic content in the lysosomal compartment, thereby clearing the cytoplasm and generating nutrients and energy. Upon autophagy initiation, cytoplasmic material becomes sequestered in newly formed double-membrane vesicles termed 'autophagosomes' that subsequently acquire acidic hydrolases for content destruction. The de novo biogenesis of autophagosomes often occurs at the endoplasmic reticulum (ER) and, in many cases, in close proximity to lipid droplets (LDs), intracellular neutral lipid storage reservoirs. LDs are targets of autophagic destruction, but have recently also been shown to contribute to autophagosome formation. In fact, some autophagy-related (Atg) proteins, such as microtubule-associated protein light chain 3 (LC3), Atg2 and Atg14L, functionally contribute to both LD and autophagosome biogenesis. In the present paper, we discuss Atg proteins, including members of the human WD-repeat protein interacting with phosphoinositides (WIPI) family that co-localize prominently with LC3, Atg2 and Atg14L to conceivably integrate LD and autophagosome dynamics.

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy.

Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca(2+)-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5-50 µM), sorafenib (1-40 µM), staurosporine (20-500 nM), or thapsigargin (20-500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca(2+) chelation by BAPTA-AM (10 and 30 µM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) α/ß and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609; 10-30 µg/ml) and 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93; 1-10 µM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca(2+) mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α(1)/α(2)-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down-regulation of AMPK by siRNA resulted in an increased basal level of both WIPI-1 puncta and LC3 lipidation, and nutrient-starvation induced autophagy was sensitive to STO-609/KN-93. Our data provide evidence that pharmacological compound-modulated and starvation-induced autophagy involves Ca(2+)-dependent signaling, including CaMKI independent of AMPKα(1)/α(2). Our data also suggest that AMPKα(1)/α(2) might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.

Starvation-induced hyperacetylation of tubulin is required for the stimulation of autophagy by nutrient deprivation.

The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.

AMPK-independent induction of autophagy by cytosolic Ca2+ increase.

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca(2+) can induce autophagy by inhibition of mTOR via the CaMKK-alpha/beta-mediated activation of AMPK. Here we demonstrate that Ca(2+) signaling can additionally induce autophagy independently of the Ca(2+)-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca(2+) induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca(2+)-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca(2+) by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca(2+)-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca(2+) flux modifications. Importantly, using AMPK-alpha1(-/-)alpha2(-/-) MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-alpha/beta inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca(2+)-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.

Assessing mammalian autophagy by WIPI-1/Atg18 puncta formation.

Macroautophagy (autophagy) is an evolutionarily highly conserved self-digestive mechanism that secures eukaryotic cellular homeostasis. Importantly, this process of intracellular bulk degradation is tightly regulated to prevent pathological consequences of disbalanced autophagic activity such as tumor development, neurodegeneration, myopathies and heart disease. A hallmark of the process of autophagy is the generation of autophagosomes, unique vesicles with double membranes of unknown origin and composition. Required for autophagosome formation is the delivery of phospholipids, such as the phosphoinositide phsophatidylinositol-3-phosphate (PI(3)P) to phagophore assembly sites (PAS). We identified the human WIPI family of 7-bladed beta-propeller proteins and found that WIPI-1 (Atg18) functions as a novel PI(3)P scaffold at the onset of autophagy. Upon binding to PI(3)P, the WIPI-1 protein accumulation at the phagophore (WIPI-1 puncta formation) can be visualized by fluorescence microscopy. Quantification of WIPI-1 puncta formation is suitable to analyze basal and induced or inhibited levels of phagophore formation, thereby assessing mammalian autophagy. Here we present an experimental step-to-step guide for assaying WIPI-1 puncta formation in human cells by confocal microscopy, live-cell imaging, and phosphoinositide binding.