Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking.

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Current methods to analyze lysosome morphology, positioning, motility and function.

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.

Thank ORP9 for FFAT: With endosomal ORP10, it's fission accomplished!

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol.https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER-endosome contact sites, with the consequent recruitment of endosomal fission factors.

Membrane trafficking: Retrofusion as an escape route out of the endosome.

Intraluminal vesicles accumulate within the endosomal lumen before lysosomal delivery or extracellular release. A new study reports the development of an elegant assay showing that these vesicles can escape from the endosomal lumen by 'back-fusion' or 'retrofusion' with the endosomal limiting membrane.

Cellular cholesterol and how to find it.

Cholesterol is an essential component of eukaryotic cellular membranes. Information about its subcellular localization and transport pathways inside cells are key for the understanding and treatment of cholesterol-related diseases. In this review we give an overview over the most commonly used methods that contributed to our current understanding of subcellular cholesterol localization and transport routes. First, we discuss methods that provide insights into cholesterol metabolism based on readouts of downstream effects such as esterification. Subsequently, we focus on the use of cholesterol-binding molecules as probes that facilitate visualization and quantification of sterols inside of cells. Finally, we explore different analogues of cholesterol which, when taken up by living cells, are integrated and transported in a similar fashion as endogenous sterols. Taken together, we highlight the challenges and advantages of each method such that researchers studying aspects of cholesterol transport may choose the most pertinent approach for their problem.

Exploiting Connections for Viral Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 (coronavirus disease 2019) pandemic, is a positive strand RNA (+RNA) virus. Like other +RNA viruses, SARS-CoV-2 is dependent on host cell metabolic machinery to survive and replicate, remodeling cellular membranes to generate sites of viral replication. Viral RNA-containing double-membrane vesicles (DMVs) are a striking feature of +RNA viral replication and are abundant in SARS-CoV-2-infected cells. Their generation involves rewiring of host lipid metabolism, including lipid biosynthetic pathways. Viruses can also redirect lipids from host cell organelles; lipid exchange at membrane contact sites, where the membranes of adjacent organelles are in close apposition, has been implicated in the replication of several +RNA viruses. Here we review current understanding of DMV biogenesis. With a focus on the exploitation of contact site machinery by +RNA viruses to generate replication organelles, we discuss evidence that similar mechanisms support SARS-CoV-2 replication, protecting its RNA from the host cell immune response.

Defective platelet function in Niemann-Pick disease type C1.

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in either NPC1 (95% of cases) or NPC2. Reduced late endosome/lysosome calcium (Ca2+) levels and the accumulation of unesterified cholesterol and sphingolipids within the late endocytic system characterize this disease. We previously reported impaired lysosome-related organelle (LRO) function in Npc1 -/- Natural Killer cells; however, the potential contribution of impaired acid compartment Ca2+ flux and LRO function in other cell types has not been determined. Here, we investigated LRO function in NPC1 disease platelets. We found elevated numbers of circulating platelets, impaired platelet aggregation and prolonged bleeding times in a murine model of NPC1 disease. Electron microscopy revealed abnormal ultrastructure in murine platelets, consistent with that seen in a U18666A (pharmacological inhibitor of NPC1) treated megakaryocyte cell line (MEG-01) exhibiting lipid storage and acidic compartment Ca2+ flux defects. Furthermore, platelets from NPC1 patients across different ages were found to cluster at the lower end of the normal range when platelet numbers were measured and had platelet volumes that were clustered at the top of the normal range. Taken together, these findings highlight the role of acid compartment Ca2+ flux in the function of platelet LROs.

Unbiased yeast screens identify cellular pathways affected in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a rare lysosomal storage disease caused by mutations in either the NPC1 or NPC2 genes. Mutations in the NPC1 gene lead to the majority of clinical cases (95%); however, the function of NPC1 remains unknown. To gain further insights into the biology of NPC1, we took advantage of the homology between the human NPC1 protein and its yeast orthologue, Niemann-Pick C-related protein 1 (Ncr1). We recreated the NCR1 mutant in yeast and performed screens to identify compensatory or redundant pathways that may be involved in NPC pathology, as well as proteins that were mislocalized in NCR1-deficient yeast. We also identified binding partners of the yeast Ncr1 orthologue. These screens identified several processes and pathways that may contribute to NPC pathogenesis. These included alterations in mitochondrial function, cytoskeleton organization, metal ion homeostasis, lipid trafficking, calcium signalling, and nutrient sensing. The mitochondrial and cytoskeletal abnormalities were validated in patient cells carrying mutations in NPC1, confirming their dysfunction in NPC disease.

Staying in touch with the endocytic network: The importance of contacts for cholesterol transport.

Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter-dependent vesicular and non-vesicular transport mechanisms. The last decade has seen a surge in interest in non-vesicular transport and inter-organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co-ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non-vesicular transport mechanisms and relationship with neurodegenerative disease.

Cholesterol Overload: Contact Sites to the Rescue!

Delivery of low-density lipoprotein-derived cholesterol to the endoplasmic reticulum (ER) is essential for cholesterol homeostasis, yet the mechanism of this transport has largely remained elusive. Two recent reports shed some light on this process, uncovering a role for Niemann Pick type-C1 protein (NPC1) in the formation of membrane contact sites (MCS) between late endosomes (LE)/lysosomes (Lys) and the ER. Both studies identified a loss of MCS in cells lacking functional NPC1, where cholesterol accumulates in late endocytic organelles. Remarkably, and taking different approaches, both studies have made a striking observation that expansion of LE/Lys-ER MCS can rescue the cholesterol accumulation phenotype in NPC1 mutant or deficient cells. In both cases, the cholesterol was shown to be transported to the ER, demonstrating the importance of ER-LE/Lys contact sites in the direct transport of low-density lipoprotein-derived cholesterol to the ER.

Roles for ER:endosome membrane contact sites in ligand-stimulated intraluminal vesicle formation.

Multivesicular endosomes/bodies (MVBs) sort membrane proteins between recycling and degradative pathways. Segregation of membrane proteins onto intraluminal vesicles (ILVs) of MVBs removes them from the recycling pathway and facilitates their degradation following fusion of MVBs with lysosomes. Sorting of many cargos onto ILVs depends on the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, although ESCRT-independent mechanisms also exist. In mammalian cells, efficient sorting of ligand-stimulated epidermal growth factor receptors onto ILVs also depends on the tyrosine phosphatase, PTP1B, an ER-localised enzyme that interacts with endosomal targets at membrane contacts between MVBs and the ER. This review focuses on the potential roles played by ER:MVB membrane contact sites in regulating ESCRT-dependent ILV formation.

Membrane dynamics and organelle biogenesis-lipid pipelines and vesicular carriers.

Discoveries spanning several decades have pointed to vital membrane lipid trafficking pathways involving both vesicular and non-vesicular carriers. But the relative contributions for distinct membrane delivery pathways in cell growth and organelle biogenesis continue to be a puzzle. This is because lipids flow from many sources and across many paths via transport vesicles, non-vesicular transfer proteins, and dynamic interactions between organelles at membrane contact sites. This forum presents our latest understanding, appreciation, and queries regarding the lipid transport mechanisms necessary to drive membrane expansion during organelle biogenesis and cell growth.

An Endosomal NAADP-Sensitive Two-Pore Ca2+ Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling.

Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca2+-dependent hubs for signaling.

Annexin A1 Tethers Membrane Contact Sites that Mediate ER to Endosome Cholesterol Transport.

Membrane contact sites between the ER and multivesicular endosomes/bodies (MVBs) play important roles in endosome positioning and fission and in neurite outgrowth. ER-MVB contacts additionally function in epidermal growth factor receptor (EGFR) tyrosine kinase downregulation by providing sites where the ER-localized phosphatase, PTP1B, interacts with endocytosed EGFR before the receptor is sorted onto intraluminal vesicles (ILVs). Here we show that these contacts are tethered by annexin A1 and its Ca(2+)-dependent ligand, S100A11, and form a subpopulation of differentially regulated contact sites between the ER and endocytic organelles. Annexin A1-regulated contacts function in the transfer of ER-derived cholesterol to the MVB when low-density lipoprotein-cholesterol in endosomes is low. This sterol traffic depends on interaction between ER-localized VAP and endosomal oxysterol-binding protein ORP1L, and is required for the formation of ILVs within the MVB and thus for the spatial regulation of EGFR signaling.

Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes.

The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt-Hoge-Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri-nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi-associated small GTPase Rab34. Rab34-positive peri-nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34-induced peri-nuclear lysosome clustering. FLCN interacts directly via its C-terminal DENN domain with the Rab34 effector RILP Using purified recombinant proteins, we show that the FLCN-DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP We propose a model whereby starvation-induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34-positive peri-nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell.

The formation and function of ER-endosome membrane contact sites.

Recent advances in membrane contact site (MCS) biology have revealed key roles for MCSs in inter-organellar exchange, the importance of which is becoming increasingly apparent. Roles for MCSs in many essential physiological processes including lipid transfer, calcium exchange, receptor tyrosine kinase signalling, lipid droplet formation, autophagosome formation, organelle dynamics and neurite outgrowth have been reported. The ER forms an extensive and dynamic network of MCSs with a diverse range of functionally distinct organelles. MCSs between the ER and endocytic pathway are particularly abundant, suggesting important physiological roles. Here, our current knowledge of the formation and function of ER contact sites with endocytic organelles from studies in mammalian systems is reviewed. Their relatively poorly defined molecular composition and recently identified functions are discussed. In addition, likely, but yet to be established, roles for these contacts in lipid transfer and calcium signalling are considered. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Coupling acidic organelles with the ER through Ca²âº microdomains at membrane contact sites.

Acidic organelles such as lysosomes serve as non-canonical Ca(2+) stores. The Ca(2+) mobilising messenger NAADP is thought to trigger local Ca(2+) release from such stores. These events are then amplified by Ca(2+) channels on canonical ER Ca(2+) stores to generate physiologically relevant global Ca(2+) signals. Coupling likely occurs at microdomains formed at membrane contact sites between acidic organelles and the ER. Molecular analyses and computational modelling suggest heterogeneity in the composition of these contacts and predicted Ca(2+) microdomain behaviour. Conversely, acidic organelles might also locally amplify and temper ER-evoked Ca(2+) signals. Ca(2+) microdomains between distinct Ca(2+) stores are thus likely to be integral to the genesis of complex Ca(2+) signals.

Methods for monitoring lysosomal morphology.

Lysosomes are abundant organelles best known for their crucial role in macromolecule turnover. Lysosome dysfunction features in several diseases exemplified by the lysosomal storage disorders and is often associated with marked changes in lysosome structure. Lysosomal morphology may therefore serve as a sensitive readout of endocytic well-being. Here we describe methods for monitoring lysosome morphology in fixed and live cells using fluorescent probes and electron microscopy.

Calcium signaling at ER membrane contact sites.

Communication between organelles is a necessary consequence of intracellular compartmentalization. Membrane contact sites (MCSs) are regions where the membranes of two organelles come into close apposition allowing exchange of small molecules and ions including Ca²âº. The ER, the cell's major Ca²âº store, forms an extensive and dynamic network of contacts with multiple organelles. Here we review established and emerging roles of ER contacts as platforms for Ca²âº exchange and further consider a potential role for Ca²âº in the regulation of MCS formation. We additionally discuss the challenges associated with the study of MCS biology and highlight advances in microscopy-based solutions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.