Transcriptional programs that control expression of the autoimmune regulator gene Aire.

Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire. In mTECs, however, Aire expression was facilitated by concurrent eviction of CTCF, specific demethylation of exon 2 and the proximal promoter, and the coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which acted on mTEC-specific accessible regions in the Aire locus.

Mitochatting - If only we could be a fly on the cell wall.

Mitochondria, cellular metabolic hubs, perform many essential processes and are required for the production of metabolites such as ATP, iron-sulfur clusters, heme, amino acids and nucleotides. To fulfill their multiple roles, mitochondria must communicate with all other organelles to exchange small molecules, ions and lipids. Since mitochondria are largely excluded from vesicular trafficking routes, they heavily rely on membrane contact sites. Contact sites are areas of close proximity between organelles that allow efficient transfer of molecules, saving the need for slow and untargeted diffusion through the cytosol. More globally, multiple metabolic pathways require coordination between mitochondria and additional organelles and mitochondrial activity affects all other cellular entities and vice versa. Therefore, uncovering the different means of mitochondrial communication will allow us a better understanding of mitochondria and may illuminate disease processes that occur in the absence of proper cross-talk. In this review we focus on how mitochondria interact with all other organelles and emphasize how this communication is essential for mitochondrial and cellular homeostasis. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

The role of iNOS in cholesterol-induced liver fibrosis.

Accumulation of cholesterol in the liver is associated with the development of non-alcoholic steatohepatitis-related fibrosis. However, underlying mechanisms are not well understood. The present study investigated the role of inducible nitric oxide synthase (iNOS) in cholesterol-induced liver fibrosis by feeding wild-type (WT) and iNOS-deficient mice with control or high-cholesterol diet (HCD) for 6 weeks. WT mice fed with HCD developed greater liver fibrosis, compared with iNOS-deficient mice, as evident by Sirius red staining and higher expression levels of profibrotic genes. Enhanced liver fibrosis in the presence of iNOS was associated with hypoxia-inducible factor-1α stabilization, matrix metalloproteinase-9 expression, and enhanced hepatic DNA damage. The profibrotic role of iNOS was also demonstrated in vivo using a selective inhibitor of iNOS as well as in vitro in a rat liver stellate cell line (HSC-T6). In conclusion, these findings suggest that iNOS is an important mediator in HCD-induced liver fibrosis.

Cnm1 mediates nucleus-mitochondria contact site formation in response to phospholipid levels.

Mitochondrial functions are tightly regulated by nuclear activity, requiring extensive communication between these organelles. One way by which organelles can communicate is through contact sites, areas of close apposition held together by tethering molecules. While many contacts have been characterized in yeast, the contact between the nucleus and mitochondria was not previously identified. Using fluorescence and electron microscopy in S. cerevisiae, we demonstrate specific areas of contact between the two organelles. Using a high-throughput screen, we uncover a role for the uncharacterized protein Ybr063c, which we have named Cnm1 (contact nucleus mitochondria 1), as a molecular tether on the nuclear membrane. We show that Cnm1 mediates contact by interacting with Tom70 on mitochondria. Moreover, Cnm1 abundance is regulated by phosphatidylcholine, enabling the coupling of phospholipid homeostasis with contact extent. The discovery of a molecular mechanism that allows mitochondrial crosstalk with the nucleus sets the ground for better understanding of mitochondrial functions in health and disease.

Metabolic alterations in the tumor microenvironment and their role in oncogenesis.

Metabolic reprogramming is a characteristic feature of both cancer cells and their neighbouring cells in the tumor microenvironment (TME). The latter include stroma fibroblasts and adipocytes, that respectively differentiate to become cancer associated fibroblasts (CAFs) and cancer associated adipocytes (CAAs), and infiltrated immune cells, that collaborate with the stromal cells to provide the tumor a pro-tumorigenic niche. Here we discuss the association between the reprogramming of glucose metabolism in the TME and oncogenic signaling and its reflection in the non-canonical functions of metabolic enzymes. We also discuss the non-canonical actions of oncometabolites and the contribution to oncogenesis of external metabolites that accumulate in the TME as result of crosstalk between the tumor and the TME. Special emphasis is given in this regard to lysophosphatidic acid (LPA) and adenosine, two powerful metabolites, the concentrations of which rise in the TME due to altered metabolism of the tumor and its surrounding cells, allowing their action as external signals.

Noncanonical regulation of phosphatidylserine metabolism by a Sec14-like protein and a lipid kinase.

The yeast phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in a membrane contact site (MCS) for PtdSer decarboxylation to phosphatidylethanolamine (PtdEtn). This proposed MCS harbors Psd2, the Sec14-like phosphatidylinositol transfer protein (PITP) Sfh4, the Stt4 phosphatidylinositol (PtdIns) 4-OH kinase, the Scs2 tether, and an uncharacterized protein. We report that, of these components, only Sfh4 and Stt4 regulate Psd2 activity in vivo. They do so via distinct mechanisms. Sfh4 operates via a mechanism for which its PtdIns-transfer activity is dispensable but requires an Sfh4-Psd2 physical interaction. The other requires Stt4-mediated production of PtdIns-4-phosphate (PtdIns4P), where Stt4 (along with the Sac1 PtdIns4P phosphatase and endoplasmic reticulum-plasma membrane tethers) indirectly modulate Psd2 activity via a PtdIns4P homeostatic mechanism that influences PtdSer accessibility to Psd2. These results identify an example in which the biological function of a Sec14-like PITP is cleanly uncoupled from its canonical in vitro PtdIns-transfer activity and challenge popular functional assumptions regarding lipid-transfer protein involvements in MCS function.

Promethin Is a Conserved Seipin Partner Protein.

Seipin (BSCL2/SPG17) is a key factor in lipid droplet (LD) biology, and its dysfunction results in severe pathologies, including the fat storage disease Berardinelli-Seip congenital lipodystrophy type 2, as well as several neurological seipinopathies. Despite its importance for human health, the molecular role of seipin is still enigmatic. Seipin is evolutionarily conserved from yeast to humans. In yeast, seipin was recently found to cooperate with the lipid droplet organization (LDO) proteins, Ldo16 and Ldo45, two structurally-related proteins involved in LD function and identity that display remote homology to the human protein promethin/TMEM159. In this study, we show that promethin is indeed an LD-associated protein that forms a complex with seipin, and its localization to the LD surface can be modulated by seipin expression levels. We thus identify promethin as a novel seipin partner protein.

The Endoplasmic Reticulum-Mitochondria Encounter Structure Complex Coordinates Coenzyme Q Biosynthesis.

Loss of the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) complex that resides in contact sites between the yeast ER and mitochondria leads to impaired respiration; however, the reason for that is not clear. We find that in ERMES null mutants, there is an increase in the level of mRNAs encoding for biosynthetic enzymes of coenzyme Q6 (CoQ6), an essential electron carrier of the mitochondrial respiratory chain. We show that the mega complexes involved in CoQ6 biosynthesis (CoQ synthomes) are destabilized in ERMES mutants. This, in turn, affects the level and distribution of CoQ6 within the cell, resulting in reduced mitochondrial CoQ6. We suggest that these outcomes contribute to the reduced respiration observed in ERMES mutants. Fluorescence microscopy experiments demonstrate close proximity between the CoQ synthome and ERMES, suggesting a spatial coordination. The involvement of the ER-mitochondria contact site in regulation of CoQ6 biogenesis highlights an additional level of communication between these two organelles.

High content image analysis reveals function of miR-124 upstream of Vimentin in regulating motor neuron mitochondria.

microRNAs (miRNAs) are critical for neuronal function and their dysregulation is repeatedly observed in neurodegenerative diseases. Here, we implemented high content image analysis for investigating the impact of several miRNAs in mouse primary motor neurons. This survey directed our attention to the neuron-specific miR-124, which controls axonal morphology. By performing next generation sequencing analysis and molecular studies, we characterized novel roles for miR-124 in control of mitochondria localization and function. We further demonstrated that the intermediate filament Vimentin is a key target of miR-124 in this system. Our data establishes a new pathway for control of mitochondria function in motor neurons, revealing the value of a neuron-specific miRNA gene as a mechanism for the re-shaping of otherwise ubiquitously-expressed intermediate filament network, upstream of mitochondria activity and cellular metabolism.

Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation.

Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus-vacuole junction contact site. In search for factors determining identity of these LDs, we screened ∼6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.

Ground control to major TOM: mitochondria-nucleus communication.

Mitochondria have crucial functions in the cell, including ATP generation, iron-sulfur cluster biogenesis, nucleotide biosynthesis, and amino acid metabolism. All of these functions require tight regulation on mitochondrial activity and homeostasis. As mitochondria biogenesis is controlled by the nucleus and almost all mitochondrial proteins are encoded by nuclear genes, a tight communication network between mitochondria and the nucleus has evolved, which includes signaling cascades, proteins which are dual-localized to the two compartments, and sensing of mitochondrial products by nuclear proteins. All of these enable a crosstalk between mitochondria and the nucleus that allows the 'ground control' to get information on mitochondria's status. Such information facilitates the creation of a cellular balance of mitochondrial status with energetic needs. This communication also allows a transcriptional response in case mitochondrial function is impaired aimed to restore mitochondrial homeostasis. As mitochondrial dysfunction is related to a growing number of genetic diseases as well as neurodegenerative conditions and aging, elucidating the mechanisms governing the mitochondrial/nuclear communication should progress a better understanding of mitochondrial dysfunctions.

A Tether Is a Tether Is a Tether: Tethering at Membrane Contact Sites.

Membrane contact sites enable interorganelle communication by positioning organelles in close proximity using molecular "tethers." With a growing understanding of the importance of contact sites, the hunt for new contact sites and their tethers is in full swing. Determining just what is a tether has proven challenging. Here, we aim to delineate guidelines that define the prerequisites for categorizing a protein as a tether. Setting this gold standard now, while groups from different disciplines are beginning to explore membrane contact sites, will enable efficient cooperation in the growing field and help to realize a great collaborative opportunity to boost its development.

Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins.

A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the mitochondrial import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in Saccharomyces cerevisiae. Cardiolipin (CL) synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4, and Om14 in mitochondria from crd1Δ cells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower in vitro capacity to import newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain de novo synthesized CL molecules. Hence, our findings suggest that CL is an important component in the biogenesis of MOM multispan proteins.

Lam6 Regulates the Extent of Contacts between Organelles.

Communication between organelles is crucial for eukaryotic cells to function as one coherent unit. An important means of communication is through membrane contact sites, where two organelles come into close proximity allowing the transport of lipids and small solutes between them. Contact sites are dynamic in size and can change in response to environmental or cellular stimuli; however, how this is regulated has been unclear. Here, we show that Saccharomyces cerevisiae Lam6 resides in several central contact sites: ERMES (ER/mitochondria encounter structure), vCLAMP (vacuole and mitochondria patch), and NVJ (nuclear vacuolar junction). We show that Lam6 is sufficient for expansion of contact sites under physiological conditions and necessary for coordination of contact site size. Given that Lam6 is part of a large protein family and is conserved in vertebrates, our work opens avenues for investigating the underlying principles of organelle communication.