Calcium storage in multivesicular endo-lysosome.

It is now established that endo-lysosomes, also referred to as late endosomes, serve as intracellular calcium store, in addition to the endoplasmic reticulum. While abundant calcium-binding proteins provide the latter compartment with its calcium storage capacity, essentially nothing is known about the mechanism responsible for calcium storage in endo-lysosomes. In this paper, we propose that the structural organization of endo-lysosomal membranes drives the calcium storage capacity of the compartment. Indeed, endo-lysosomes exhibit a characteristic multivesicular ultrastructure, with intralumenal membranes providing a large amount of additional bilayer surface. We used a theoretical approach to investigate the calcium storage capacity of endosomes, using known calcium binding affinities for bilayers and morphological data on endo-lysosome membrane organization. Finally, we tested our predictions experimentally after Sorting Nexin 3 depletion to decrease the intralumenal membrane content. We conclude that the major negatively-charge lipids and proteins of endo-lysosomes serve as calcium-binding molecules in the acidic calcium stores of mammalian cells, while the large surface area of intralumenal membranes provide the necessary storage capacity.

Life in the lumen: The multivesicular endosome.

The late endosomes/endo-lysosomes of vertebrates contain an atypical phospholipid, lysobisphosphatidic acid (LBPA) (also termed bis[monoacylglycero]phosphate [BMP]), which is not detected elsewhere in the cell. LBPA is abundant in the membrane system present in the lumen of this compartment, including intralumenal vesicles (ILVs). In this review, the current knowledge on LBPA and LBPA-containing membranes will be summarized, and their role in the control of endosomal cholesterol will be outlined. Some speculations will also be made on how this system may be overwhelmed in the cholesterol storage disorder Niemann-Pick C. Then, the roles of intralumenal membranes in endo-lysosomal dynamics and functions will be discussed in broader terms. Likewise, the mechanisms that drive the biogenesis of intralumenal membranes, including ESCRTs, will also be discussed, as well as their diverse composition and fate, including degradation in lysosomes and secretion as exosomes. This review will also discuss how intralumenal membranes are hijacked by pathogenic agents during intoxication and infection, and what is the biochemical composition and function of the intra-endosomal lumenal milieu. Finally, this review will allude to the size limitations imposed on intralumenal vesicle functions and speculate on the possible role of LBPA as calcium chelator in the acidic calcium stores of endo-lysosomes.

Drug-induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann-Pick type C cells and mice.

Most cells acquire cholesterol by endocytosis of circulating low-density lipoproteins (LDLs). After cholesteryl ester de-esterification in endosomes, free cholesterol is redistributed to intracellular membranes via unclear mechanisms. Our previous work suggested that the unconventional phospholipid lysobisphosphatidic acid (LBPA) may play a role in modulating the cholesterol flux through endosomes. In this study, we used the Prestwick library of FDA-approved compounds in a high-content, image-based screen of the endosomal lipids, lysobisphosphatidic acid and LDL-derived cholesterol. We report that thioperamide maleate, an inverse agonist of the histamine H3 receptor HRH3, increases highly selectively the levels of lysobisphosphatidic acid, without affecting any endosomal protein or function that we tested. Our data also show that thioperamide significantly reduces the endosome cholesterol overload in fibroblasts from patients with the cholesterol storage disorder Niemann-Pick type C (NPC), as well as in liver of Npc1-/- mice. We conclude that LBPA controls endosomal cholesterol mobilization and export to cellular destinations, perhaps by fluidifying or buffering cholesterol in endosomal membranes, and that thioperamide has repurposing potential for the treatment of NPC.

Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

The topology of the lymphotoxin ß receptor that accumulates upon endolysosomal dysfunction dictates the NF-κB signaling outcome.

Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells.

In specialized cell types, lysosome-related organelles support regulated secretory pathways, whereas in nonspecialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol-storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in nonspecialized cells and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel mucolipin-1, as well as FYCO1, the AP1 adaptor, and its partner Gadkin. We conclude that endo-lysosomes in nonspecialized cells can acquire secretory functions elicited by cyclodextrin and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes.

L-leucyl-L-leucine methyl ester (LLOMe) induces apoptosis, which is thought to be mediated by release of lysosomal cysteine cathepsins from permeabilized lysosomes into the cytosol. Here, we demonstrated in HeLa cells that apoptotic as well as sub-apoptotic concentrations of LLOMe caused rapid and complete lysosomal membrane permeabilization (LMP), as evidenced by loss of the proton gradient and release into the cytosol of internalized lysosomal markers below a relative molecular mass of 10,000. However, there was no evidence for the release of cysteine cathepsins B and L into the cytosol; rather they remained within lysosomes, where they were rapidly inactivated and degraded. LLOMe-induced adverse effects, including LMP, loss of cysteine cathepsin activity, caspase activation and cell death could be reduced by inhibition of cathepsin C, but not by inhibiting cathepsins B and L. When incubated with sub-apoptotic LLOMe concentrations, lysosomes transiently lost protons but annealed and re-acidified within hours. Full lysosomal function required new protein synthesis of cysteine cathepsins and other hydrolyses. Our data argue against the release of lysosomal enzymes into the cytosol and their proposed proteolytic signaling during LLOMe-induced apoptosis.

Wnt directs the endosomal flux of LDL-derived cholesterol and lipid droplet homeostasis.

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

Endosome maturation, transport and functions.

Efficient sorting of the material internalized by endocytosis is essential for key cellular functions and represents a, if not the, major trafficking pathway in mammalian cells. Incoming material - solutes, receptors and cargos, lipids and even pathogenic agents - are routed to various destinations within mammalian cells at two major sorting stations: the early and late endosome. The early endosome receives all manner of incoming material from the plasma membrane, as well as from the Golgi, and serves as an initial sorting nexus routing molecules back to the cell surface through recycling endosomes, to the trans-Golgi network by retrograde transport, or on to the late endosome/lysosome. The early endosome also regulates cell signaling, through the downregulation of internalized receptors, which are packaged into intralumenal vesicles that arise from inward invaginations of the limiting membrane. These multivesicular regions detach or mature from early endosomes and become free endocytic carrier vesicle/multivesicular body, which transports cargoes to late endosomes. The late endosome provides a central hub for incoming traffic from the endocytic, biosynthetic and autophagic pathways and outgoing traffic to the lysosomes, the Golgi complex or the plasma membrane. They also function as a key sensing/signaling platform that inform the cell about the nutrient situation. Herein we summarize the current understanding of the organization and functions of the endocytic pathway, differences across species, and the process of endosome maturation.

Selective Imaging of Late Endosomes with a pH-Sensitive Diazaoxatriangulene Fluorescent Probe.

Late endosomes are a major trafficking hub in the cell at the crossroads between endocytosis, autophagy, and degradation in lysosomes. Herein is disclosed the first small molecule allowing their selective imaging and monitoring in the form of a diazaoxatriangulene fluorophore, 1a (hexadecyl side chain). The compound is prepared in three steps from a simple carbenium precursor. In nanospheres, this pH-sensitive (pKa = 7.3), photochemically stable dye fluoresces in the red part of visible light (601 and 578 nm, acid and basic forms, respectively) with a quantum yield between 14 and 16% and an excited-state lifetime of 7.7-7.8 ns. Importantly, the protonated form 1a·H(+) provokes a specific staining of late endosome compartments (pH 5.0-5.5) after 5 h of incubation with HeLa cells. Not surprisingly, this late endosome marking depends on the intra-organelle pH, and changing the nature of the lipophilic chain provokes a loss of selectivity. Interestingly, fixation of the fluorophore is readily achieved with paraformaldehyde, giving the possibility to image both live and fixed cells.

Automated Microscopy and High Content Screens (Phenotypic Screens) in Academia Labs.

Imaged-based screening has been developing extremely quickly in the past 10 years. Academic institutes quickly realized that the discovery capacity of this technology was huge, allowing the automatic detection and quantification of complex cell phenotypes. Associated with chemical or genetic perturbations, high content screening is the method of choice for a deep system biology analysis. The evolution of high-content screening is mainly due to the recent progress in the development of fast and high quality automated imagers and of a plethora of new very bright fluorescent markers, so that almost any cellular element can be seen and imaged. In this paper we review and summarize the major steps in the development of an image-based screening project.

Regulation of the MVB pathway by SCAMP3.

The biogenesis of multivesicular endosomes and the sorting of activated signaling receptors into multivesicular endosomes depend on soluble protein complexes (ESCRT complexes), which transiently interact with the receptor cargo and the endosomal membrane. Previously, it was shown that the transmembrane protein secretory carrier membrane protein (SCAMP) 3, which is present on endosomes, interacts with ESCRT components. Here, we report that SCAMP3 plays a role in the biogenesis of multivesicular endosomes. We find that SCAMP3 plays a role in EGF receptor sorting into multivesicular endosomes and in the formation of intralumenal vesicles within these endosomes in vitro and thus also controls EGF receptor targeting to lysosomes. We also find that SCAMP3 regulates the EGF-dependent biogenesis of multivesicular endosomes. We conclude that the transmembrane protein SCAMP3 has a positive role in sorting into and budding of intralumenal vesicles and thereby controls the process of multivesicular endosome biogenesis.

Discovery of novel membrane binding structures and functions.

The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms and could uncover novel sites for therapeutic intervention. We present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH), and PX domains bind membranes, the resulting membrane optimal docking area (MODA) method yields predictions for a given protein of known three-dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain, and Neisseria gonorrhoeae MsrB protein and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR), which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible and provides a new tool for functional annotation of the proteome.

PNPLA3/adiponutrin functions in lipid droplet formation.

BACKGROUND INFORMATION: In animals, adipose tissue contains the main energy store as lipid droplets (LDs) composed of esterified cholesterol (CE) and triacylglycerol (TAG) enveloped in a mono-layer of phospholipid and decorated by a coat of proteins. Upon increased energy demand, dedicated lipases hydrolyse TAG stepwise into free fatty acids that are released in circulation and made available to peripheral tissue. In case of aberrant caloric load, TAGs are deposited into non-adipocyte tissues, primarily liver cells. For instance, non-alcoholic fatty liver disease (NAFLD) is a common chronic disorder characterised by an excess of TAG in the liver of patients regardless of their susceptibility to obesity, diabetes or exposure to alcohol. Several independent linkage studies have associated NAFLD with a non-synonymous variant of patatin-like phospholipase domain-containing 3 (PNPLA3/adiponutrin) encoding an isoleucine to methionine substitution at position 148 (I148M) (see Cohen et al., 2011 for review). However, the mechanism by which a variation in PNPLA3 gives susceptibility to NAFLD is not known, primarily because the physiological role of PNPLA3 still needs to be elucidated. RESULTS: We have identified PNPLA3 in a screen for genes upregulated by intracellular lipid accumulation. We investigated the sub-cellular distribution and potential function of PNPLA3 in fibroblast-like cells supplemented with lipids. We demonstrate that PNPLA3 is targetted to LDs in a process that requires an intact Brummer box domain, which is conserved in the patatin-like phospholipase family. We show that increased levels of the NAFLD-linked PNPLA3 isoform leads to larger LDs, whereas decreased levels of PNPLA3 had the opposite effect. Interestingly, however, PNPLA3 induced a reduction in LD size upon co-expression with ABDH5/CGI-58, an activator of the TAG lipase PNPLA2, which is the closest homolog of PNPLA3. By investigating LD populations according to their size and composition, we show that perturbing intracellular lipid trafficking drastically modifies LD nature. CONCLUSIONS: Taken together, our results suggest that PNPLA3 exhibits a dual function in LD metabolism, and that it participates in the restoration of lipid homeostasis upon aberrant intracellular lipid accumulation.

Single-cell analysis of population context advances RNAi screening at multiple levels.

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.

Ion flux and the function of endosomes and lysosomes: pH is just the start: the flux of ions across endosomal membranes influences endosome function not only through regulation of the luminal pH.

The ionic nature of endosomes varies considerably in character along the endocytic pathway. Counter-ion flux across the limiting membrane of endosomes has long been considered essential for full acidification and normal endosome/lysosomal function. The proximal functions of luminal ions, however, have been difficult to assess. The recent development of transgenic mice carrying mutations in the intracellular chloride channels (ClCs) has provided a tool to uncouple Cl(-) influx from endosomal acidification. Intriguingly, many of the defects of the endo-lysomal system attributed to aberrant pH persist in the Cl(-)-deficient mice implying a direct regulatory role for Cl(-) influx in endosome function. These observations may begin to explain the abundance of endosomal ion transporters, including ClCs, sodium-proton exchangers, two-pore channels and mucolipins, that have been localized to endo-lysosomes, and the extensive changes in luminal ion composition therein. In this review, we summarize what is known regarding the mediators of endosomal ion flux, and discuss the implications of changing ionic content on endo-lysosomal function.

Nanoparticle entry into cells; the cell biology weak link.

Nanoparticles (NP) are attractive options for the therapeutic delivery of active pharmaceutical drugs, proteins and nucleic acids into cells, tissues and organs. Research into the development and application of NP most often starts with a diverse group of scientists, including chemists, bioengineers and material and pharmaceutical scientists, who design, fabricate and characterize NP in vitro (Stage 1). The next step (Stage 2) generally investigates cell toxicity as well as the processes by which NP bind, are internalized and deliver their cargo to appropriate model tissue culture cells. Subsequently, in Stage 3, selected NP are tested in animal systems, mostly mouse. Whereas the chemistry-based development and analysis in Stage 1 is increasingly sophisticated, the investigations in Stage 2 are not what could be regarded as 'state-of-the-art' for the cell biology field and the quality of research into NP interactions with cells is often sub-standard. In this review we describe our current understanding of the mechanisms by which particles gain entry into mammalian cells via endocytosis. We summarize the most important areas for concern, highlight some of the most common mis-conceptions, and identify areas where NP scientists could engage with trained cell biologists. Our survey of the different mechanisms of uptake into cells makes us suspect that claims for roles for caveolae, as well as macropinocytosis, in NP uptake into cells have been exaggerated, whereas phagocytosis has been under-appreciated.

Lipids in endocytic membrane transport and sorting.

Protein complexes associated to specific membrane lipids and protein-lipid domains contribute to regulate protein sorting and membrane dynamics in the endocytic pathway. It is also becoming apparent that different lipid territories are distributed along the pathway, and that some lipids segregate into specialised microdomains.

Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies.

After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs--an adaptor--like protein that binds membrane PtdIns3P via a FYVE motif-and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.

A novel strategy to identify drugs that interfere with endosomal lipids.

Lipids are major components of the cell and, like proteins, exhibit much diversity and are highly regulated. And yet, our knowledge of lipids remains limited primarily because their study is difficult. We will use novel Systems Biology approaches, and in particular high content screening techniques, to investigate the mechanisms that regulate the cellular lipid content and function. Our project is to carry out a small compound screen using lipid imaging techniques to identify conditions that interfere with cellular levels and distribution of cholesterol, lysobisphosphatic acid and phosphoinositol-3-phosphate. This forward chemical genetic screen approach should reveal new molecular tools to investigate the molecular mechanism involved in the regulation of these lipids. The aim is to apply chemical proteomic techniques to identify the molecular target(s) of compounds able to affect the intracellular cholesterol regulation and to assess if these are novel druggable targets. This will be the ideal complementary study to the RNAi screen, currently run in our group, as the effect of the inhibition caused by a small molecule can be rapidly reversed when this is removed. Such a small molecule can be administered to a cell or an animal for a very short time to study the function of the target protein and to look at biological mechanisms in a short time-frame. This project is highly interdisciplinary, and will benefit from the help of the screening core facility, currently developed with the support of the NCCR.