Inter-organelle Membrane Contact Sites Research Articles

Endo-lysosomal dysfunction and neuronal-glial crosstalk in Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a rare progressive lysosomal lipid storage disorder that manifests with a heterogeneous spectrum of clinical syndromes, including visceral, neurological and psychiatric symptoms. This monogenetic autosomal recessive disease is largely caused by mutations in the NPC1 gene, which controls intracellular lipid homeostasis. Vesicle-mediated endo-lysosomal lipid trafficking and non-vesicular lipid exchange via inter-organelle membrane contact sites are both regulated by the NPC1 protein. Loss of NPC1 function therefore triggers intracellular accumulation of diverse lipid species, including cholesterol, glycosphingolipids, sphingomyelin and sphingosine. The NPC1-mediated dysfunction of lipid transport has severe consequences for all brain cells, leading to neurodegeneration. Besides the cell-autonomous contribution of neuronal NPC1, aberrant NPC1 signalling in other brain cells is critical for the pathology. We discuss here the importance of endo-lysosomal dysfunction and a tight crosstalk between neurons, oligodendrocytes, astrocytes and microglia in NPC pathology. We strongly believe that a cell-specific rescue may not be sufficient to counteract the severity of the NPC pathology, but targeting common mechanisms, such as endo-lysosomal and lipid trafficking dysfunction, may ameliorate NPC pathology. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Phosphoinositides in New Spaces.

Phosphoinositides (PIs) are phospholipids derived from phosphatidylinositol. PIs are regulated via reversible phosphorylation, which is directed by the opposing actions of PI kinases and phosphatases. PIs constitute a minor fraction of the total cellular lipid pool but play pleiotropic roles in multiple aspects of cell biology. Genetic mutations of PI regulatory enzymes have been identified in rare congenital developmental syndromes, including ciliopathies, and in numerous human diseases, such as cancer and metabolic and neurological disorders. Accordingly, PI regulatory enzymes have been targeted in the design of potential therapeutic interventions for human diseases. Recent advances place PIs as central regulators of membrane dynamics within functionally distinct subcellular compartments. This brief review focuses on the emerging role PIs play in regulating cell signaling within the primary cilium and in directing transfer of molecules at interorganelle membrane contact sites and identifies new roles for PIs in subcellular spaces.

Liver inter-organelle membrane contact sites revealed by serial section electron tomography.

Inter-organelle membrane contact sites (MCSs) are defined as areas of close proximity between the membranes of two organelles (10-80nm). They have been implicated in many physiological processes such as Ca++, lipids or small molecules transfer, organelles biogenesis or dynamic and have an important role in many cellular processes such as apoptosis, autophagy, and signaling. Since the distance and the extent of these contacts are in the nanometer range, high resolution techniques are ideal for imaging these structures. It is for this reason that transmission electron microscopy (TEM) has been considered the gold standard for MCSs visualization and the first technique that described them. However, often TEM analysis is limited to 2D lacking information on the 3D association between the organelles involved in MCSs. To fully describe the complex architecture of MSCs and to unveil their role in cellular physiology a 3D analysis is required. This chapter provides a method for the analysis of MCSs using serial section electron tomography (ssET), a technique able to reconstruct in 3D at nanometer resolution cellular and subcellular structures. By applying this procedure, it was possible to elucidate the role of the contacts between Endoplasmic Reticulum (ER) and other organelles in liver lipid metabolism.

Super-Resolution Microscopy to Study Interorganelle Contact Sites

Interorganelle membrane contact sites (MCS) are areas of close vicinity between the membranes of two organelles that are maintained by protein tethers. Recently, a significant research effort has been made to study MCS, as they are implicated in a wide range of biological functions, such as organelle biogenesis and division, apoptosis, autophagy, and ion and phospholipid homeostasis. Their composition, characteristics, and dynamics can be studied by different techniques, but in recent years super-resolution fluorescence microscopy (SRFM) has emerged as a powerful tool for studying MCS. In this review, we first explore the main characteristics and biological functions of MCS and summarize the different approaches for studying them. Then, we center on SRFM techniques that have been used to study MCS. For each of the approaches, we summarize their working principle, discuss their advantages and limitations, and explore the main discoveries they have uncovered in the field of MCS.

Alterations in Lysosome Homeostasis in Lipid-Related Disorders: Impact on Metabolic Tissues and Immune Cells.

Lipid-related disorders, which primarily affect metabolic tissues, including adipose tissue and the liver are associated with alterations in lysosome homeostasis. Obesity is one of the more prevalent diseases, which results in energy imbalance within metabolic tissues and lysosome dysfunction. Less frequent diseases include Niemann-Pick type C (NPC) and Gaucher diseases, both of which are known as Lysosomal Storage Diseases (LSDs), where lysosomal dysfunction within metabolic tissues remains to be fully characterized. Adipocytes and hepatocytes share common pathways involved in the lysosome-autophagic axis, which are regulated by the function of cathepsins and CD36, an immuno-metabolic receptor and display alterations in lipid diseases, and thereby impacting metabolic functions. In addition to intrinsic defects observed in metabolic tissues, cells of the immune system, such as B cells can infiltrate adipose and liver tissues, during metabolic imbalance favoring inflammation. Moreover, B cells rely on lysosomes to promote the processing and presentation of extracellular antigens and thus could also present lysosome dysfunction, consequently affecting such functions. On the other hand, growing evidence suggests that cells accumulating lipids display defective inter-organelle membrane contact sites (MCSs) established by lysosomes and other compartments, which contribute to metabolic dysfunctions at the cellular level. Overall, in this review we will discuss recent findings addressing common mechanisms that are involved in lysosome dysregulation in adipocytes and hepatocytes during obesity, NPC, and Gaucher diseases. We will discuss whether these mechanisms may modulate the function of B cells and how inter-organelle contacts, emerging as relevant cellular mechanisms in the control of lipid homeostasis, have an impact on these diseases.

Quantifying inter-organelle membrane contact sites using proximity ligation assay in fixed optic nerve sections

Membrane contact sites (MCS) play crucial roles in cell physiology with dysfunction in several MCS proteins being linked with neurological and optic nerve diseases. Although there have been significant advances in imaging these interactions over the past two decades with advanced electron microscopy techniques, super-resolution imaging and proximity-dependent fluorescent reporters, a technique to observe and quantify MCS in mammalian optic nerve tissues has not yet been reported. We demonstrate for the first time that proximity ligation assay (PLA), a technique already used in mammalian cell lines, can be used as an efficient method of quantifying inter-organelle contact sites, namely mitochondria-endoplasmic reticulum (ER) and mitochondria-late-endosomes, in mammalian optic nerve tissues treated with adeno-associated virus (AAV) gene therapy with wild-type or phosphomimetic (active) protrudin. PLA utilises complementary single-stranded DNA oligomers bound to secondary antibodies that hybridise and complete a circular piece of DNA when the primary antibodies of interest interact. These interactions can be detected by amplifying the circular DNA and adding fluorescent probes. We show that PLA is a useful method that can be used to quantify MCS in optic nerve tissues. We have found that upregulation of protrudin with gene therapy significantly increases the number of mitochondria-ER and mitochondria-Rab7-late endosomes contact sites in optic nerves.

Lipid Droplets as Regulators of Metabolism and Immunity

Abstract A hallmark of sterile and nonsterile inflammation is the increased accumulation of cytoplasmic lipid droplets (LDs) in non-adipose cells. LDs are ubiquitous organelles specialized in neutral lipid storage and hydrolysis. Originating in the ER, LDs are comprised of a core of neutral lipids (cholesterol esters, triglycerides) surrounded by a phospholipid monolayer and several LD-associated proteins. The perilipin (PLIN1-5) family are the most abundant structural proteins present on the surface of LDs. While PLIN1 is primarily expressed in adipocytes, PLIN2 and PLIN3 are ubiquitously expressed. LDs also acquire a host of enzymes and proteins that regulate LD metabolism. Amongst these are neutral lipases and selective lipophagy factors that promote hydrolysis of LD-associated neutral lipid. In addition, LDs physically associate with other organelles such as mitochondria through inter-organelle membrane contact sites that facilitate lipid transport. Beyond serving as a source of energy storage, LDs participate in inflammatory and infectious diseases, regulating both innate and adaptive host immune responses. Here, we review recent studies on the role of LDs in the regulation of immunometabolism.

Mitochondria-lysosome membrane contacts are defective in GDAP1-related Charcot-Marie-Tooth disease.

Mutations in the GDAP1 gene cause Charcot–Marie–Tooth (CMT) neuropathy. GDAP1 is an atypical glutathione S-transferase (GST) of the outer mitochondrial membrane and the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs). Here, we investigate the role of this GST in the autophagic flux and the membrane contact sites (MCSs) between mitochondria and lysosomes in the cellular pathophysiology of GDAP1 deficiency. We demonstrate that GDAP1 participates in basal autophagy and that its depletion affects LC3 and PI3P biology in autophagosome biogenesis and membrane trafficking from MAMs. GDAP1 also contributes to the maturation of lysosome by interacting with PYKfyve kinase, a pH-dependent master lysosomal regulator. GDAP1 deficiency causes giant lysosomes with hydrolytic activity, a delay in the autophagic lysosome reformation, and TFEB activation. Notably, we found that GDAP1 interacts with LAMP-1, which supports that GDAP1–LAMP-1 is a new tethering pair of mitochondria and lysosome membrane contacts. We observed mitochondria–lysosome MCSs in soma and axons of cultured mouse embryonic motor neurons and human neuroblastoma cells. GDAP1 deficiency reduces the MCSs between these organelles, causes mitochondrial network abnormalities, and decreases levels of cellular glutathione (GSH). The supply of GSH-MEE suffices to rescue the lysosome membranes and the defects of the mitochondrial network, but not the interorganelle MCSs nor early autophagic events. Overall, we show that GDAP1 enables the proper function of mitochondrial MCSs in both degradative and nondegradative pathways, which could explain primary insults in GDAP1-related CMT pathophysiology, and highlights new redox-sensitive targets in axonopathies where mitochondria and lysosomes are involved.

Current and Emerging Approaches for Studying Inter-Organelle Membrane Contact Sites

Inter-organelle membrane contact sites (MCSs) are classically defined as areas of close proximity between heterologous membranes and established by specific proteins (termed tethers). The interest on MCSs has rapidly increased in the last years, since MCSs play a crucial role in the transfer of cellular components between different organelles and have been involved in important cellular functions such as apoptosis, organelle division and biogenesis, and cell growth. Recently, an unprecedented depth and breadth in insights into the details of MCSs have been uncovered. On one hand, extensive MCSs (organelles interactome) are revealed by comprehensive analysis of organelle network with high temporal-spatial resolution at the system level. On the other hand, more and more tethers involving in MCSs are identified and further works are focusing on addressing the role of these tethers in regulating the function of MCSs at the molecular level. These enormous progresses largely depend on the powerful approaches, including several different types of microscopies and various biochemical techniques. These approaches have greatly accelerated recent advances in MCSs at the system and molecular level. In this review, we summarize the current and emerging approaches for studying MCSs, such as various microscopies, proximity-driven fluorescent signal generation and proximity-dependent biotinylation. In addition, we highlight the advantages and disadvantages of the techniques to provide a general guidance for the study of MCSs.

Inter-Organelle Membrane Contact Sites and Mitochondrial Quality Control during Aging: A Geroscience View.

Mitochondrial dysfunction and failing mitochondrial quality control (MQC) are major determinants of aging. Far from being standalone organelles, mitochondria are intricately related with cellular other compartments, including lysosomes. The intimate relationship between mitochondria and lysosomes is reflected by the fact that lysosomal degradation of dysfunctional mitochondria is the final step of mitophagy. Inter-organelle membrane contact sites also allow bidirectional communication between mitochondria and lysosomes as part of nondegradative pathways. This interaction establishes a functional unit that regulates metabolic signaling, mitochondrial dynamics, and, hence, MQC. Contacts of mitochondria with the endoplasmic reticulum (ER) have also been described. ER-mitochondrial interactions are relevant to Ca2+ homeostasis, transfer of phospholipid precursors to mitochondria, and integration of apoptotic signaling. Many proteins involved in mitochondrial contact sites with other organelles also participate to degradative MQC pathways. Hence, a comprehensive assessment of mitochondrial dysfunction during aging requires a thorough evaluation of degradative and nondegradative inter-organelle pathways. Here, we present a geroscience overview on (1) degradative MQC pathways, (2) nondegradative processes involving inter-organelle tethering, (3) age-related changes in inter-organelle degradative and nondegradative pathways, and (4) relevance of MQC failure to inflammaging and age-related conditions, with a focus on Parkinson’s disease as a prototypical geroscience condition.

Roles of protrudin at interorganelle membrane contact sites.

Intracellular organelles were long viewed as isolated compartments floating in the cytosol. However, this view has been radically changed within the last decade by the discovery that most organelles communicate with the endoplasmic reticulum (ER) network via membrane contact sites (MCSs) that are essential for intracellular homeostasis. Protrudin is an ER resident protein that was originally shown to regulate neurite formation by promoting endosome trafficking. More recently, however, protrudin has been found to serve as a tethering factor at MCSs. The roles performed by protrudin at MCSs are mediated by its various domains, including inactivation of the small GTPase Rab11, bending of the ER membrane, and functional interactions with other molecules such as the motor protein KIF5 and the ER protein VAP. Mutations in the protrudin gene (ZFYVE27) are associated with hereditary spastic paraplegia, an axonopathy that results from defective ER structure. This review, examines the pleiotropic molecular functions of protrudin and its role in interorganellar communication.

Regulation and Function of Mitochondria-Lysosome Membrane Contact Sites in Cellular Homeostasis.

Mitochondrial and lysosomal function are intricately related and critical for maintaining cellular homeostasis, as highlighted by multiple diseases linked to dysfunction of both organelles. Recent work using high-resolution microscopy demonstrates the dynamic formation of inter-organelle membrane contact sites between mitochondria and lysosomes, allowing for their direct interaction in a pathway distinct from mitophagy or lysosomal degradation of mitochondrial-derived vesicles. Mitochondria-lysosome contact site tethering is mechanistically regulated by mitochondrial proteins promoting Rab7 GTP hydrolysis, and allows for the bidirectional crosstalk between mitochondria and lysosomes and the regulation of their organelle network dynamics, including mitochondrial fission. In this review, we summarize recent advances in mitochondria-lysosome contact site regulation and function, and discuss their potential roles in cellular homeostasis and various human diseases.

A Third Musketeer on the ER: MOSPD2 is a Novel VAP-related Receptor for FFAT Motifs

Interorganelle membrane contact sites are subcellular structures that favor exchange and communication inside the cell. Such microdomains are built by molecular bridges that create a physical connection between two distinct organelles. The field of contact sites is now flourishing with discoveries of new tethering molecules. In that context, we identified by an unbiased proteomic approach a novel scaffold protein named MOtile SPerm Domain-containing protein 2 (MOSPD2). MOSPD2 is an endoplasmic reticulum (ER)-resident protein that is able to interact with several organelle-bound proteins that possess a small motif, named FFAT (two phenylalanines in an acidic tract). Consequently, we showed that MOSPD2 and its protein partners build contacts between the ER and endosomes, mitochondria, or Golgi. These findings highlight a new way for docking organelles on the ER.

Regulation of Endoplasmic Reticulum-Mitochondria Ca2+ Transfer and Its Importance for Anti-Cancer Therapies.

Inter-organelle membrane contact sites are emerging as major sites for the regulation of intracellular Ca2+ concentration and distribution. Here, extracellular stimuli operate on a wide array of channels, pumps, and ion exchangers to redistribute intracellular Ca2+ among several compartments. The resulting highly defined spatial and temporal patterns of Ca2+ movement can be used to elicit specific cellular responses, including cell proliferation, migration, or death. Plasma membrane (PM) also can directly contact mitochondria and endoplasmic reticulum (ER) through caveolae, small invaginations of the PM that ensure inter-organelle contacts, and can contribute to the regulation of numerous cellular functions through scaffolding proteins such as caveolins. PM and ER organize specialized junctions. Here, many components of the receptor-dependent Ca2+ signals are clustered, including the ORAI1-stromal interaction molecule 1 complex. This complex constitutes a primary mechanism for Ca2+ entry into non-excitable cells, modulated by intracellular Ca2+. Several contact sites between the ER and mitochondria, termed mitochondria-associated membranes, show a very complex and specialized structure and host a wide number of proteins that regulate Ca2+ transfer. In this review, we summarize current knowledge of the particular action of several oncogenes and tumor suppressors at these specialized check points and analyze anti-cancer therapies that specifically target Ca2+ flow at the inter-organelle contacts to alter the metabolism and fate of the cancer cell.

Discovery and Roles of ER-Endolysosomal Contact Sites in Disease.

Inter-organelle membrane contact sites (MCSs) serve as unique microenvironments for the sensing and exchange of cellular metabolites and lipids. Though poorly defined, ER-endolysosomal contact sites are quickly becoming recognized as centers for inter-organelle lipid exchange and metabolic decision-making. Here, we review the discovery and current state of knowledge of ER-endolysosomal MCSs with particular focus on the molecular players that establish and/or utilize these contact sites in metabolism. We also discuss associations of ER-endolysosomal MCS-associated proteins in human disease, as well as the therapeutic promise these contact sites hold in modulating cellular physiology.

Organelle remodeling at membrane contact sites.

Cellular organelles must execute sophisticated biological processes to persist, and often communicate with one another to exchange metabolites and information. Recent studies suggest inter-organelle membrane contact sites (MCSs) are hubs for this cellular cross-talk. MCSs also govern membrane remodeling, thus controlling aspects of organelle shape, identity, and function. Here, we summarize three emerging phenomena that MCSs appear to govern: 1) organelle identity via the non-vesicular exchange of lipids, 2) mitochondrial shape and division, and 3) endosomal migration in response to sterol trafficking. We also discuss the role for ER-endolysosomal contact sites in cholesterol metabolism, and the potential biomedical importance this holds. Indeed, the emerging field inter-organellar cross-talk promises substantial advances in the fields of lipid metabolism and cell signaling.

ACBD2/ECI2-Mediated Peroxisome-Mitochondria Interactions in Leydig Cell Steroid Biosynthesis.

Fatty acid metabolism and steroid biosynthesis are 2 major pathways shared by peroxisomes and mitochondria. Both organelles are in close apposition to the endoplasmic reticulum, with which they communicate via interorganelle membrane contact sites to promote cellular signaling and the exchange of ions and lipids. To date, no convincing evidence of the direct contact between peroxisomes and mitochondria was reported in mammalian cells. Hormone-induced, tightly controlled steroid hormone biosynthesis requires interorganelle interactions. Using immunofluorescent staining and live-cell imaging, we found that dibutyryl-cAMP treatment of MA-10 mouse tumor Leydig cells rapidly induces peroxisomes to approach mitochondria and form peroxisome-mitochondrial contact sites/fusion, revealed by the subcellular distribution of the endogenous acyl-coenzyme A-binding domain (ACBD)2/ECI2 isoform A generated by alternative splicing, and further validated using a proximity ligation assay. This event occurs likely via a peroxisome-like structure, which is mediated by peroxisomal and mitochondrial matrix protein import complexes: peroxisomal import receptor peroxisomal biogenesis factor 5 (PEX5), and the mitochondrial import receptor subunit translocase of outer mitochondrial membrane 20 homolog (yeast) protein. Similar results were obtained using the mLTC-1 mouse tumor Leydig cells. Ectopic expression of the ACBD2/ECI2 isoform A in MA-10 cells led to increased basal and hormone-stimulated steroid formation, indicating that ACBD2/ECI2-mediated peroxisomes-mitochondria interactions favor in the exchange of metabolites and/or macromolecules between these 2 organelles in support of steroid biosynthesis. Considering the widespread occurrence of the ACBD2/ECI2 protein, we propose that this protein might serve as a tool to assist in understanding the contact between peroxisomes and mitochondria.