Inter-organelle Contact Sites Research Articles

A novel bacterial effector protein mediates ER-LD membrane contacts to regulate host lipid droplets.

Effective intracellular communication between cellular organelles occurs at dedicated membrane contact sites (MCSs). Tether proteins are responsible for the establishment of MCSs, enabling direct communication between organelles to ensure organelle function and host cell homeostasis. While recent research has identified tether proteins in several bacterial pathogens, their functions have predominantly been associated with mediating inter-organelle communication between the bacteria containing vacuole (BCV) and the host endoplasmic reticulum (ER). Here, we identify a novel bacterial effector protein, CbEPF1, which acts as a molecular tether beyond the confines of the BCV and facilitates interactions between host cell organelles. Coxiella burnetii, an obligate intracellular bacterial pathogen, encodes the FFAT motif-containing protein CbEPF1 which localizes to host lipid droplets (LDs). CbEPF1 establishes inter-organelle contact sites between host LDs and the ER through its interactions with VAP family proteins. Intriguingly, CbEPF1 modulates growth of host LDs in a FFAT motif-dependent manner. These findings highlight the potential for bacterial effector proteins to impact host cellular homeostasis by manipulating inter-organelle communication beyond conventional BCVs.

Rab18 Drift in Lipid Droplet and Endoplasmic Reticulum Interactions of Adipocytes under Obesogenic Conditions.

The adipose tissue stores excess energy in the form of neutral lipids within adipocyte lipid droplets (LDs). The correct function of LDs requires the interaction with other organelles, such as the endoplasmic reticulum (ER) as well as with LD coat-associated proteins, including Rab18, a mediator of intracellular lipid trafficking and ER-LD interaction. Although perturbations of the inter-organelle contact sites have been linked to several diseases, such as cancer, no information regarding ER-LD contact sites in dysfunctional adipocytes from the obese adipose tissue has been published to date. Herein, the ER-LD connection and Rab18 distribution at ER-LD contact sites are examined in adipocytes challenged with fibrosis and inflammatory conditions, which represent known hallmarks of the adipose tissue in obesity. Our results show that adipocytes differentiated in fibrotic conditions caused ER fragmentation, the expansion of ER-LD contact sites, and modified Rab18 dynamics. Likewise, adipocytes exposed to inflammatory conditions favored ER-LD contact, Rab18 accumulation in the ER, and Rab18 redistribution to large LDs. Finally, our studies in human adipocytes supported the suggestion that Rab18 transitions to the LD coat from the ER. Taken together, our results suggest that obesity-related pathogenic processes alter the maintenance of ER-LD interactions and interfere with Rab18 trafficking through these contact sites.

Misregulation of mitochondria-lysosome contact dynamics in Charcot-Marie-Tooth Type 2B disease Rab7 mutant sensory peripheral neurons.

Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.

Perturbation of the host cell Ca2+ homeostasis and ER-mitochondria contact sites by the SARS-CoV-2 structural proteins E and M

Coronavirus disease (COVID-19) is a contagious respiratory disease caused by the SARS-CoV-2 virus. The clinical phenotypes are variable, ranging from spontaneous recovery to serious illness and death. On March 2020, a global COVID-19 pandemic was declared by the World Health Organization (WHO). As of February 2023, almost 670 million cases and 6,8 million deaths have been confirmed worldwide. Coronaviruses, including SARS-CoV-2, contain a single-stranded RNA genome enclosed in a viral capsid consisting of four structural proteins: the nucleocapsid (N) protein, in the ribonucleoprotein core, the spike (S) protein, the envelope (E) protein, and the membrane (M) protein, embedded in the surface envelope. In particular, the E protein is a poorly characterized viroporin with high identity amongst all the β-coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43) and a low mutation rate. Here, we focused our attention on the study of SARS-CoV-2 E and M proteins, and we found a general perturbation of the host cell calcium (Ca2+) homeostasis and a selective rearrangement of the interorganelle contact sites. In vitro and in vivo biochemical analyses revealed that the binding of specific nanobodies to soluble regions of SARS-CoV-2 E protein reversed the observed phenotypes, suggesting that the E protein might be an important therapeutic candidate not only for vaccine development, but also for the clinical management of COVID designing drug regimens that, so far, are very limited.

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy.

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

Filling in the gaps: SNX-RGS proteins as multiorganelle tethers.

SNX-RGS proteins are molecular tethers localized to multiple interorganelle contact sites that exhibit roles in cellular metabolism. Here, we highlight recent findings on these proteins and discuss their emerging roles in metabolism, human disease, and lipid trafficking.

Circulating Mitochondrial DNA and Inter-Organelle Contact Sites in Aging and Associated Conditions.

Mitochondria are primarily involved in cell bioenergetics, regulation of redox homeostasis, and cell death/survival signaling. An immunostimulatory property of mitochondria has also been recognized which is deployed through the extracellular release of entire or portioned organelle and/or mitochondrial DNA (mtDNA) unloading. Dynamic homo- and heterotypic interactions involving mitochondria have been described. Each type of connection has functional implications that eventually optimize mitochondrial activity according to the bioenergetic demands of a specific cell/tissue. Inter-organelle communications may also serve as molecular platforms for the extracellular release of mitochondrial components and subsequent ignition of systemic inflammation. Age-related chronic inflammation (inflamm-aging) has been associated with mitochondrial dysfunction and increased extracellular release of mitochondrial components—in particular, cell-free mtDNA. The close relationship between mitochondrial dysfunction and cellular senescence further supports the central role of mitochondria in the aging process and its related conditions. Here, we provide an overview of (1) the mitochondrial genetic system and the potential routes for generating and releasing mtDNA intermediates; (2) the pro-inflammatory pathways elicited by circulating mtDNA; (3) the participation of inter-organelle contacts to mtDNA homeostasis; and (4) the link of these processes with senescence and age-associated conditions.

Get Closer to the World of Contact Sites: A Beginner's Guide to Proximity-Driven Fluorescent Probes.

To maintain cellular homeostasis and to coordinate the proper response to a specific stimulus, information must be integrated throughout the cell in a well-organized network, in which organelles are the crucial nodes and membrane contact sites are the main edges. Membrane contact sites are the cellular subdomains where two or more organelles come into close apposition and interact with each other. Even though many inter-organelle contacts have been identified, most of them are still not fully characterized, therefore their study is an appealing and expanding field of research. Thanks to significant technological progress, many tools are now available or are in rapid development, making it difficult to choose which one is the most suitable for answering a specific biological question. Here we distinguish two different experimental approaches for studying inter-organelle contact sites. The first one aims to morphologically characterize the sites of membrane contact and to identify the molecular players involved, relying mainly on the application of biochemical and electron microscopy (EM)-related methods. The second approach aims to understand the functional importance of a specific contact, focusing on spatio-temporal details. For this purpose, proximity-driven fluorescent probes are the experimental tools of choice, since they allow the monitoring and quantification of membrane contact sites and their dynamics in living cells under different cellular conditions or upon different stimuli. In this review, we focus on these tools with the purpose of highlighting their great versatility and how they can be applied in the study of membrane contacts. We will extensively describe all the different types of proximity-driven fluorescent tools, discussing their benefits and drawbacks, ultimately providing some suggestions to choose and apply the appropriate methods on a case-to-case basis and to obtain the best experimental outcomes.

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 Droplet-Organelle Contact Sites as Hubs for Fatty Acid Metabolism, Trafficking, and Metabolic Channeling.

Cells prepare for fluctuations in nutrient availability by storing energy in the form of neutral lipids in organelles called Lipid Droplets (LDs). Upon starvation, fatty acids (FAs) released from LDs are trafficked to different cellular compartments to be utilized for membrane biogenesis or as a source of energy. Despite the biochemical pathways being known in detail, the spatio-temporal regulation of FA synthesis, storage, release, and breakdown is not completely understood. Recent studies suggest that FA trafficking and metabolism are facilitated by inter-organelle contact sites that form between LDs and other cellular compartments such as the Endoplasmic Reticulum (ER), mitochondria, peroxisomes, and lysosomes. LD-LD contact sites are also sites where FAs are transferred in a directional manner to support LD growth and expansion. As the storage site of neutral lipids, LDs play a central role in FA homeostasis. In this mini review, we highlight the role of LD contact sites with other organelles in FA trafficking, channeling, and metabolism and discuss the implications for these pathways on cellular lipid and energy homeostasis.

Partitioning of the mevalonate synthesizing enzymes at ER‐lysosome contacts influences their catalytic efficiency

The last decade of molecular biology is hallmarked by an extraordinary increase in our understanding of previously unappreciated spatial scales in biology. Enzymes and metabolites are partitioned into distinct membrane-bound organelles to spatially regulate metabolism and signaling. More recent discoveries have uncovered that enzymes can also inducibly or permanently compartmentalize within sub-organelle domains such as inter-organelle contact sites (1). However, a key knowledge gap is understanding role that enzyme partitioning plays on regulating enzyme function or pathway flux. Here, we performed a GFP-based screen in Saccharomyces cerevisiae to identify metabolic enzymes that may enrich at sub-organelle domains. We found that during Acute Glucose Restriction (AGR) the rate-limiting enzymes of sterol biogenesis, Hmg1 and Hmg2, partition at the Nucleus-Vacuole Junction (NVJ), an inter-organelle contact site between the nuclear envelope and yeast vacuole. NVJ partitioning of Hmg1/2 correlates with increased de-novo synthesis of sterol-esters. Additionally, Hmg1/2 partitioning can be blocked by deletion of the NVJ tether protein Nvj1. Remarkably, Hmg1/2 is less catalytically efficient in Nvj1 knock-out cells, suggesting a potential link between its spatial compartmentalization and increased enzymatic activity. We further demonstrate that Nvj1-mediated partitioning of Hmg1/2 during AGR contributes to cellular fitness, and coincides with ability to resume growth following glucose starvation. Overall, we have uncovered a previously underappreciated level of spatial regulation for Hmg1/2, and also identified a novel role for inter-organelle contact sites in the regulation of mevalonate metabolism.

Improved Split-GFP Systems for Visualizing Organelle Contact Sites in Yeast and Human Cells.

Inter-organelle contact sites have attracted a lot of attention as functionally specialized regions that mediate the exchange of metabolites, including lipids and ions, between distinct organelles. However, studies on inter-organelle contact sites are at an early stage and it remains enigmatic what directly mediates the organelle-organelle interactions and how the number and degree of the contacts are regulated. As a first step to answer these questions, we previously developed split-GFP probes that could visualize and quantify multiple inter-organelle contact sites in the yeast and human cultured cells. However, the split-GFP probes possessed a disadvantage of inducing artificial connections between two different organelle membranes, especially when overexpressed. In the present study, we developed a way to express the split-GFP probes whose expressions remained at low levels, with minimal variations between different yeast cells. Besides, we constructed a HeLa cell line in which the expression of the split-GFP probes could be induced by the addition of doxycycline to minimize the artificial effects. The improved split-GFP systems may be faithful tools to quantify organelle contact sites and screen new factors involved in organelle-organelle tethering in yeast and mammalian cells.

MICOS subcomplexes assemble independently on the mitochondrial inner membrane in proximity to ER contact sites

MICOS is a conserved multisubunit complex that localizes to mitochondrial cristae junctions and organizes cristae positioning within the organelle. MICOS is organized into two independent subcomplexes; however, the mechanisms that dictate the assembly and spatial positioning of each MICOS subcomplex are poorly understood. Here, we determine that MICOS subcomplexes target independently of one another to sites on the inner mitochondrial membrane that are in proximity to contact sites between mitochondria and the ER. One subcomplex, composed of Mic27/Mic26/Mic10/Mic12, requires ERMES complex function for its assembly. In contrast, the principal MICOS component, Mic60, self-assembles and localizes in close proximity to the ER through an independent mechanism. We also find that Mic60 can uniquely redistribute adjacent to forced mitochondria-vacuole contact sites. Our data suggest that nonoverlapping properties of interorganelle contact sites provide spatial cues that enable MICOS assembly and ultimately lead to proper physical and functional organization of mitochondria.

Lipid homeostasis in mitochondria.

Mitochondria are surrounded by the two membranes, the outer and inner membranes, whose lipid compositions are optimized for proper functions and structural organizations of mitochondria. Although a part of mitochondrial lipids including their characteristic lipids, phosphatidylethanolamine and cardiolipin, are synthesized within mitochondria, their precursor lipids and other lipids are transported from other organelles, mainly the ER. Mitochondrially synthesized lipids are re-distributed within mitochondria and to other organelles, as well. Recent studies pointed to the important roles of inter-organelle contact sites in lipid trafficking between different organelle membranes. Identification of Ups/PRELI proteins as lipid transfer proteins shuttling between the mitochondrial outer and inner membranes established a part of the molecular and structural basis of the still elusive intra-mitochondrial lipid trafficking.

Nucleus–Plasma Membrane Contact Sites Are Formed During Spermiogenesis in the Acoel Symsagittifera roscoffensis

Symsagittifera roscoffensis is a small marine worm found in the intertidal zone of sandy beaches around the European shores of the Atlantic. S. roscoffensis is a member of the Acoelomorpha, a group of flatworms formerly classified with the Platyhelminthes, but now recognized as Xenacoelomorpha, a separate phylum of disputed affinity. We have used electron microscopy to examine the process of spermiogenesis (the final stage of spermatogenesis) in S. roscoffensis, by which spermatids form highly elongated spermatozoa. Their nuclei are long and thread-like, running most of the cell’s length, and during the process, a pair of flagella are fully incorporated into the cell body. Two previously undescribed interorganelle contact sites form at different stages of spermiogenesis. Strikingly, there is an extensive nucleus–plasma membrane contact site. Golgi-derived granules containing electron-dense filaments line up along the spermatid plasma membrane, undergo a conformational change, and donate material that forms a perinuclear layer that cements this contact site. We also show in spermatids at an earlier stage that the same granules are associated with microtubules, presumably for traffic along the elongating cell. We identify a second spermiogenesis-specific contact site where sheaths engulfing each internalizing flagellum contact the nuclear envelope.

Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system

Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.

Direct Detection of ER-mitochondrial Contacts with Fully Quantified Fluorescence Microscopy

Inter-organelle contacts between the endoplasmic reticulum and the mitochondria are tightly regulated in cells. ER-mitochondrial contact sites have been painstakingly characterized through biochemical means, and yet much remains to be understood due to their small physical extent. As a result, even with super-resolution imaging, these inter-organelle contact sites are difficult to study through fluorescence colocalization measurements. Here, we have developed Fully Quantified Fluorescence Microscopy for use with nearly any fluorescence microscope. With this, we are working to characterize the spatial and temporal properties of ER-mitochondrial contact sites in live cells.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

The conserved GTPase Gem1 regulates endoplasmic reticulum–mitochondria connections

Mitochondria are connected to the endoplasmic reticulum (ER) through specialized protein complexes. We recently identified the ER-mitochondria encounter structure (ERMES) tethering complex, which plays a role in phospholipid exchange between the two organelles. ERMES also has been implicated in the coordination of mitochondrial protein import, mitochondrial DNA replication, and mitochondrial dynamics, suggesting that these interorganelle contact sites play central regulatory roles in coordinating various aspects of the physiology of the two organelles. Here we purified ERMES complexes and identified the Ca(2+)-binding Miro GTPase Gem1 as an integral component of ERMES. Gem1 regulates the number and size of the ERMES complexes. In vivo, association of Gem1 to ERMES required the first of Gem1's two GTPase domains and the first of its two functional Ca(2+)-binding domains. In contrast, Gem1's second GTPase domain was required for proper ERMES function in phospholipid exchange. Our results suggest that ERMES is not a passive conduit for interorganellar lipid exchange, but that it can be regulated in response to physiological needs. Furthermore, we provide evidence that the metazoan Gem1 ortholog Miro-1 localizes to sites of ER-mitochondrial contact, suggesting that some of the features ascribed to Gem1 may be evolutionarily conserved.