The ORP9-ORP11 dimer promotes sphingomyelin synthesis.

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.

Daily Light Onset and Plasma Membrane Tethers Regulate Mitochondria Redistribution within the Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is an essential component of the retina that plays multiple roles required to support visual function. These include light onset- and circadian rhythm-dependent tasks, such as daily phagocytosis of photoreceptor outer segments. Mitochondria provide energy to the highly specialized and energy-dependent RPE. In this study, we examined the positioning of mitochondria and how this is influenced by the onset of light. We identified a population of mitochondria that are tethered to the basal plasma membrane pre- and post-light onset. Following light onset, mitochondria redistributed apically and interacted with melanosomes and phagosomes. In a choroideremia mouse model that has regions of the RPE with disrupted or lost infolding of the plasma membrane, the positionings of only the non-tethered mitochondria were affected. This provides evidence that the tethering of mitochondria to the plasma membrane plays an important role that is maintained under these disease conditions. Our work shows that there are subpopulations of RPE mitochondria based on their positioning after light onset. It is likely they play distinct roles in the RPE that are needed to fulfil the changing cellular demands throughout the day.

Exploring the pathological mechanisms underlying Cohen syndrome.

Cohen Syndrome (CS) is a rare autosomal recessive disorder caused by biallelic mutations in the VPS13B gene. It is characterized by multiple clinical features, including acquired microcephaly, developmental delay, intellectual disability, neutropenia, and retinal degeneration. VPS13B is part of the bridge-like lipid transport (BLTP) protein family, which in mammals also includes VPS13A, -C, and -D. The proteins of this family are peripheral membrane proteins with different sub-cellular localization, but all share similar structural features and have been proposed to act as lipid transport proteins at organellar membrane contact sites. VPS13B is localized at the Golgi apparatus and is essential for the maintenance of organelle architecture. Here we present a review of the experimental data on the function of the protein at the cellular level, discussing the potential link with disease phenotype and review the studies on animal models recapitulating features of the human disease.

Proximity driven plastid-nucleus relationships are facilitated by tandem plastid-ER dynamics.

Peri-nuclear clustering (PNC) of chloroplasts has largely been described in senescent and pathogen- or ROS- stressed cells. Stromules, tubular plastid extensions are also observed under similar conditions. Coincident observations of PNC and stromules associate the two phenomena in facilitating retrograde signaling between chloroplasts and the nucleus. However, PNC incidence in non-stressed cells under normal growth and developmental conditions, when stromules are usually not observed, remains unclear. Using transgenic Arabidopsis expressing different organelle-targeted fluorescent proteins we show that PNC is a dynamic subcellular phenomenon that continues in the absence of light and is not dependent on stromule formation. PNC is facilitated by tandem plastid-ER dynamics created through membrane contact sites between the two organelles. While PNC increases upon ER-membrane expansion, some plastids may remain in the peri-nuclear region due to their localization in ER-lined nuclear indentions. Moreover, some PNC plastids may sporadically extend stromules into ER-lined nuclear grooves. Our findings strongly suggest that PNC is not an exclusive response to stress caused by pathogens, high light or exogenous-H2O2 treatment and does not require stromule formation. However, morphological and behavioural alterations in ER and concomitant changes in tandem, plastid-ER dynamics play a major role in facilitating the phenomenon.

Calcium channel signalling at neuronal endoplasmic reticulum-plasma membrane junctions.

Neurons are highly specialised cells that need to relay information over long distances and integrate signals from thousands of synaptic inputs. The complexity of neuronal function is evident in the morphology of their plasma membrane (PM), by far the most intricate of all cell types. Yet, within the neuron lies an organelle whose architecture adds another level to this morphological sophistication - the endoplasmic reticulum (ER). Neuronal ER is abundant in the cell body and extends to distant axonal terminals and postsynaptic dendritic spines. It also adopts specialised structures like the spine apparatus in the postsynapse and the cisternal organelle in the axon initial segment. At membrane contact sites (MCSs) between the ER and the PM, the two membranes come in close proximity to create hubs of lipid exchange and Ca2+ signalling called ER-PM junctions. The development of electron and light microscopy techniques extended our knowledge on the physiological relevance of ER-PM MCSs. Equally important was the identification of ER and PM partners that interact in these junctions, most notably the STIM-ORAI and VAP-Kv2.1 pairs. The physiological functions of ER-PM junctions in neurons are being increasingly explored, but their molecular composition and the role in the dynamics of Ca2+ signalling are less clear. This review aims to outline the current state of research on the topic of neuronal ER-PM contacts. Specifically, we will summarise the involvement of different classes of Ca2+ channels in these junctions, discuss their role in neuronal development and neuropathology and propose directions for further research.

Building a biofactory: Constructing glandular trichomes in Cannabis sativa.

Flowers of Cannabis sativa L. are densely covered with glandular trichomes containing cannabis resin that is used for medicinal and recreational purposes. The highly productive glandular trichomes have been described as 'biofactories.' In this review, we use this analogy to highlight recent advances in cannabis cell biology, metabolomics, and transcriptomics. The biofactory is built by epidermal outgrowths that differentiate into peltate-like glandular trichome heads, consisting of a disc of interconnected secretory cells with unique cellular structures. Cannabinoid and terpenoid products are warehoused in the extracellular storage cavity. Finally, multicellular stalks raise the glandular heads above the epidermis, giving cannabis flower their frosty appearance.

Spartin is a Lipid Transfer Protein That Facilitates Lipid Droplet Turnover.

One means by which cells reutilize neutral lipids stored in lipid droplets is to degrade them by autophagy. This process involves spartin, mutations of which cause the rare inherited disorder Troyer syndrome (or spastic paraplegia-20, SPG20). A recently published paper from the team led by Karin Reinsich (Yale) suggests that the molecular function of spartin and its unique highly conserved "senescence" domain is as a lipid transfer protein. Spartin binds to and transfers all lipid species found in lipid droplets, from phospholipids to triglycerides and sterol esters. This lipid transfer activity correlates with spartin's ability to sustain lipid droplet turnover. The senescence domain poses an intriguing question around the wide range of its cargoes, but intriguingly it has yet to yield up its secrets because attempts at crystallization failed and AlphaFold's prediction is unconvincing.

Unraveling the Connection: Cholesterol, Calcium Signaling, and Neurodegeneration.

Cholesterol and calcium play crucial roles as integral structural components and functional signaling entities within the central nervous system. Disruption in cholesterol homeostasis has been linked to Alzheimer's, Parkinson's, and Huntington's Disease while alterations in calcium signaling is hypothesized to be a key substrate for neurodegeneration across many disorders. Despite the importance of regulated cholesterol and calcium homeostasis for brain health there has been an absence of research investigating the interdependence of these signaling molecules and how they can tune each other's abundance at membranes to influence membrane identity. Here, we discuss the role of cholesterol in shaping calcium dynamics in a neurodegenerative disorder that arises due to mutations in the lysosomal cholesterol transporter, Niemann Pick Type C1 (NPC1). We discuss the molecular mechanisms through which altered lysosomal cholesterol transport influences calcium signaling pathways through remodeling of ion channel distribution at organelle-organelle membrane contacts leading to neurodegeneration. This scientific inquiry not only sheds light on NPC disease but also holds implications for comprehending other cholesterol-associated neurodegenerative disorders.

YdbH and YnbE form an intermembrane bridge to maintain lipid homeostasis in the outer membrane of Escherichia coli.

The outer membrane (OM) of didermic gram-negative bacteria is essential for growth, maintenance of cellular integrity, and innate resistance to many antimicrobials. Its asymmetric lipid distribution, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet, is required for these functions. Lpt proteins form a transenvelope bridge that transports newly synthesized LPS from the inner membrane (IM) to OM, but how the bulk of phospholipids are transported between these membranes is poorly understood. Recently, three members of the AsmA-like protein family, TamB, YhdP, and YdbH, were shown to be functionally redundant and were proposed to transport phospholipids between IM and OM in Escherichia coli. These proteins belong to the repeating ß-groove superfamily, which includes eukaryotic lipid-transfer proteins that mediate phospholipid transport between organelles at contact sites. Here, we show that the IM-anchored YdbH protein interacts with the OM lipoprotein YnbE to form a functional protein bridge between the IM and OM in E. coli. Based on AlphaFold-Multimer predictions, genetic data, and in vivo site-directed cross-linking, we propose that YnbE interacts with YdbH through ß-strand augmentation to extend the continuous hydrophobic ß-groove of YdbH that is thought to shield acyl chains of phospholipids as they travel through the aqueous intermembrane periplasmic compartment. Our data also suggest that the periplasmic protein YdbL prevents extensive amyloid-like multimerization of YnbE in cells. We, therefore, propose that YdbL has a chaperone-like function that prevents uncontrolled runaway multimerization of YnbE to ensure the proper formation of the YdbH-YnbE intermembrane bridge.

Non-vesicular phosphatidylinositol transfer plays critical roles in defining organelle lipid composition.

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.

Altered GM1 catabolism affects NMDAR-mediated Ca2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model.

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to ß-galactosidase deficiency drastically alters neuronal Ca2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated N-methyl D-aspartate receptor (NMDAR) Ca2+ channel, thereby increasing Ca2+ flux, activating extracellular signal-regulated kinase (ERK) signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and worsens the generalized neuronal cell death characteristic of GM1-gangliosidosis.

OSBP-mediated PI(4)P-cholesterol exchange at endoplasmic reticulum-secretory granule contact sites controls insulin secretion.

Insulin is packaged into secretory granules that depart the Golgi and undergo a maturation process that involves changes in the protein and lipid composition of the granules. Here, we show that insulin secretory granules form physical contacts with the endoplasmic reticulum and that the lipid exchange protein oxysterol-binding protein (OSBP) is recruited to these sites in a Ca2+-dependent manner. OSBP binding to insulin granules is positively regulated by phosphatidylinositol-4 (PI4)-kinases and negatively regulated by the PI4 phosphate (PI(4)P) phosphatase Sac2. Loss of Sac2 results in excess accumulation of cholesterol on insulin granules that is normalized when OSBP expression is reduced, and both acute inhibition and small interfering RNA (siRNA)-mediated knockdown of OSBP suppress glucose-stimulated insulin secretion without affecting insulin production or intracellular Ca2+ signaling. In conclusion, we show that lipid exchange at endoplasmic reticulum (ER)-granule contact sites is involved in the exocytic process and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation.

Membrane contact sites regulate vacuolar fission via sphingolipid metabolism.

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus-vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions.

Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.

Plastid Transient and Stable Interactions with Other Cell Compartments.

Plastids are organelles delineated by two envelopes playing important roles in different cellular processes such as energy production or lipid biosynthesis. To regulate their biogenesis and their function, plastids have to communicate with other cellular compartments. This communication can be mediated by metabolites, signaling molecules, and by the establishment of direct contacts between the plastid envelope and other organelles such as the endoplasmic reticulum, mitochondria, peroxisomes, plasma membrane, and the nucleus. These interactions are highly dynamic and respond to different biotic and abiotic stresses. However, the mechanisms involved in the formation of plastid-organelle contact sites and their functions are still far from being understood. In this chapter, we summarize our current knowledge about plastid contact sites and their role in the regulation of plastid biogenesis and function.

Analysis of a Super-Complex at Contact Sites Between Mitochondria and Plastids.

Plastids are organelles playing fundamental roles in different cellular processes such as energy metabolism or lipid biosynthesis. To fulfill their biogenesis and their function in the cell, plastids have to communicate with other cellular compartments. This communication can be mediated by the establishment of direct contact sites between plastids envelop and other organelles. These contacts are dynamic structures regulated in response to stress. For example, during phosphate (Pi) starvation, the number of contact sites between plastids and mitochondria significantly increases. In this situation, these contacts play an important role in the transfer of galactoglycerolipids from plastids to mitochondria. Recently, Pi starvation stress was used to identify key proteins involved in the traffic of galactoglycerolipids from plastids to mitochondria in Arabidopsis thaliana. A mitochondrial lipoprotein complex called MTL (Mitochondrial Transmembrane Lipoprotein) was identified. This complex contains mitochondrial proteins but also proteins located in the plastid envelope, suggesting its presence at the plastid-mitochondria junction. This chapter describes the protocol to isolate the MTL complex by clear-native polyacrylamide gel electrophoresis (CN-PAGE) from the mitochondrial fraction of Arabidopsis cell cultures and the methods to study different features of this complex.

The interactions of subcellular organelles in pulmonary fibrosis induced by carbon black nanoparticles: a comprehensive review.

Owing to the widespread use and improper emissions of carbon black nanoparticles (CBNPs), the adverse effects of CBNPs on human health have attracted much attention. In toxicological research, carbon black is frequently utilized as a negative control because of its low toxicity and poor solubility. However, recent studies have indicated that inhalation exposure to CBNPs could be a risk factor for severe and prolonged pulmonary inflammation and fibrosis. At present, the pathogenesis of pulmonary fibrosis induced by CBNPs is still not fully elucidated, but it is known that with small particle size and large surface area, CBNPs are more easily ingested by cells, leading to organelle damage and abnormal interactions between organelles. Damaged organelle and abnormal organelles interactions lead to cell structure and function disorders, which is one of the important factors in the development and occurrence of various diseases, including pulmonary fibrosis. This review offers a comprehensive analysis of organelle structure, function, and interaction mechanisms, while also summarizing the research advancements in organelles and organelle interactions in CBNPs-induced pulmonary fibrosis.

OSBP is a Major Determinant of Golgi Phosphatidylinositol 4-Phosphate Homeostasis.

The lipid phosphatidylinositol 4-phosphate (PI4P) plays a master regulatory role at Golgi membranes, orchestrating membrane budding, non-vesicular lipid transport and membrane organization. It follows that harmonious Golgi function requires strictly maintained PI4P homeostasis. One of the most abundant PI4P effector proteins is the oxysterol binding protein (OSBP), a lipid transfer protein that exchanges trans-Golgi PI4P for ER cholesterol. Although this protein consumes PI4P as part of its lipid anti-porter function, whether it actively contributes to Golgi PI4P homeostasis has been questioned. Here, we employed a series of acute and chronic genetic manipulations, together with orthogonal targeting of OSBP, to interrogate its control over Golgi PI4P abundance. Modulating OSBP levels at ER:Golgi membrane contact sites produces reciprocal changes in PI4P levels. Additionally, we observe that OSBP has a high capacity for PI4P turnover, even at orthogonal organelle membranes. However, despite also visiting the plasma membrane, endogenous OSBP makes no impact on PI4P levels in this compartment. We conclude that OSBP is a major determinant of Golgi PI4P homeostasis.

Contact-FP: A Dimerization-Dependent Fluorescent Protein Toolkit for Visualizing Membrane Contact Site Dynamics.

Membrane contact sites (MCSs) are sites of close apposition between two organelles used to exchange ions, lipids, and information. Cells respond to changing environmental or developmental conditions by modulating the number, extent, or duration of MCSs. Because of their small size and dynamic nature, tools to study the dynamics of MCSs in live cells have been limited. Dimerization-dependent fluorescent proteins (ddFPs) targeted to organelle membranes are an ideal tool for studying MCS dynamics because they reversibly interact to fluoresce specifically at the interface between two organelles. Here, we build on previous work using ddFPs as sensors to visualize the morphology and dynamics of MCSs. We engineered a suite of ddFPs called Contact-FP that targets ddFP monomers to lipid droplets (LDs), the endoplasmic reticulum (ER), mitochondria, peroxisomes, lysosomes, plasma membrane, caveolae, and the cytoplasm. We show that these probes correctly localize to their target organelles. Using LDs as a test case, we demonstrate that Contact-FP pairs specifically localize to the interface between two target organelles. Titration of LD-mitochondria ddFPs revealed that these sensors can be used at high concentrations to drive MCSs or can be titrated down to minimally perturb and visualize endogenous MCSs. We show that Contact-FP probes can be used to: (1) visualize LD-mitochondria MCS dynamics, (2) observe changes in LD-mitochondria MCS dynamics upon overexpression of PLIN5, a known LD-mitochondrial tether, and (3) visualize two MCSs that share one organelle simultaneously (e.g., LD-mitochondria and LD-ER MCSs). Contact-FP probes can be optimized to visualize MCSs between any pair of organelles represented in the toolkit.

Remodelling of mitochondrial function by import of specific lipids at multiple membrane-contact sites.

Organelles form physical and functional contact between each other to exchange information, metabolic intermediates, and signaling molecules. Tethering factors and contact site complexes bring partnering organelles into close spatial proximity to establish membrane contact sites (MCSs), which specialize in unique functions like lipid transport or Ca2+ signaling. Here, we discuss how MCSs form dynamic platforms that are important for lipid metabolism. We provide a perspective on how import of specific lipids from the ER and other organelles may contribute to remodeling of mitochondria during nutrient starvation. We speculate that mitochondrial adaptation is achieved by connecting several compartments into a highly dynamic organelle network. The lipid droplet appears to be a central hub in coordinating the function of these organelle neighborhoods.