Anti-apoptotic MCL-1 promotes long-chain fatty acid oxidation through interaction with ACSL1.

MCL-1 is essential for promoting the survival of many normal cell lineages and confers survival and chemoresistance in cancer. Beyond apoptosis regulation, MCL-1 has been linked to modulating mitochondrial metabolism, but the mechanism(s) by which it does so are unclear. Here, we show in tissues and cells that MCL-1 supports essential steps in long-chain (but not short-chain) fatty acid ß-oxidation (FAO) through its binding to specific long-chain acyl-coenzyme A (CoA) synthetases of the ACSL family. ACSL1 binds to the BH3-binding hydrophobic groove of MCL-1 through a non-conventional BH3-domain. Perturbation of this interaction, via genetic loss of Mcl1, mutagenesis, or use of selective BH3-mimetic MCL-1 inhibitors, represses long-chain FAO in cells and in mouse livers and hearts. Our findings reveal how anti-apoptotic MCL-1 facilitates mitochondrial metabolism and indicate that disruption of this function may be associated with unanticipated cardiac toxicities of MCL-1 inhibitors in clinical trials.

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen-Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2.

Monoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood1. Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders2. Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission3-5. VMATs are also therapeutic drug targets for a number of different conditions6-9. Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin. Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access. Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.

Lipid droplets in the nervous system.

Lipid droplets are dynamic intracellular lipid storage organelles that respond to the physiological state of cells. In addition to controlling cell metabolism, they play a protective role for many cellular stressors, including oxidative stress. Despite prior descriptions of lipid droplets appearing in the brain as early as a century ago, only recently has the role of lipid droplets in cells found in the brain begun to be understood. Lipid droplet functions have now been described for cells of the nervous system in the context of development, aging, and an increasing number of neuropathologies. Here, we review the basic mechanisms of lipid droplet formation, turnover, and function and discuss how these mechanisms enable lipid droplets to function in different cell types of the nervous system under healthy and pathological conditions.

ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

A Study of Opiate, Opiate Metabolites and Antihistamines in Urine after Consumption of Cold Syrups by LC-MS/MS.

Studying the origin of opiate and/or opiate metabolites in individual urine specimens after consumption of cold syrups is vital for patients, doctors, and law enforcement. A rapid liquid chromatography-tandem mass spectrometry method using "dilute-and-shoot" analysis without the need for extraction, hydrolysis and/or derivatization has been developed and validated. The approach provides linear ranges of 2.5-1000 ng mL-1 for 6-acetylmorphine, codeine, chlorpheniramine, and carbinoxamine, 2.5-800 ng mL-1 for morphine and morphine-3-ß-d-glucuronide, and 2.5-600 ng mL-1 for morphine-6-ß-d-glucuronide and codeine-6-ß-d-glucuronide, with excellent correlation coefficients (R2 > 0.995) and matrix effects (< 5%). Urine samples collected from the ten participants orally administered cold syrups were analyzed. The results concluded that participants consuming codeine-containing cold syrups did not routinely pass urine tests for opiates, and their morphine-codeine concentration ratios (M/C) were not always < 1. In addition, the distribution map of the clinical total concentration of the sum of morphine and codeine against the antihistamines (chlorpheniramine or carbinoxamine) were plotted for discrimination of people who used cold syrups. The 15 real cases have been studied by using M/C rule, cutoff value, and distribution map, further revealing a potential approach to determine opiate metabolite in urine originating from cold syrups.

Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

A Photoluminescent Colorimetric Probe of Bovine Serum Albumin-Stabilized Gold Nanoclusters for New Psychoactive Substances: Cathinone Drugs in Seized Street Samples.

Screening of illicit drugs for new psychoactive substances-namely cathinone-at crime scenes is in high demand. A dual-emission bovine serum albumin-stabilized gold nanoclusters probe was synthesized and used for quantitation and screening of 4-chloromethcathinone and cathinone analogues in an aqueous solution. The photoluminescent (PL) color of the bovine serum albumin-stabilized Au nanoclusters (BSA-Au NCs) probe solution changed from red to dark blue during the identification of cathinone drugs when excited using a portable ultraviolet light-emitting diodes lamp (365 nm). This probe solution allows the PL color-changing point and limit of detection down to 10.0 and 0.14 mM, respectively, for 4-chloromethcathinone. The phenomenon of PL color-changing of BSA-Au NCs was attributed to its PL band at 650 nm, quenching through an electron transfer mechanism. The probe solution was highly specific to cathinone drugs, over other popular illicit drugs, including heroin, cocaine, ketamine, and methamphetamine. The practicality of this BSA-Au NCs probe was assessed by using it to screen illicit drugs seized by law enforcement officers. All 20 actual cases from street and smuggling samples were validated using this BSA-Au NCs probe solution and then confirmed using gas chromatography-mass spectrometry. The results reveal this BSA-Au NCs probe solution is practical for screening cathinone drugs at crime scenes.

Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III.

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two interrelated mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1- and CHMP1B-dependent modifications in LD membrane morphology. Furthermore, LD-to-peroxisome FA trafficking mediated by M1 Spastin is required to relieve LDs of lipid peroxidation. M1 Spastin's dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may underlie the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity.

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial ß-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-Stimulated Live Cells.

Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM) important for Ca2+ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P2 homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM junctions, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to monitor PI(4,5)P2 replenishment following receptor stimulation, as a readout of PI transfer, using a PI(4,5)P2 biosensor and total internal reflection fluorescence microscopy. The second protocol directly visualizes PI transfer proteins that accumulate at ER-PM junctions and mediate PI(4,5)P2 replenishment with PI in the ER in stimulated cells. These methods provide spatial and temporal analysis of ER-to-PM PI transfer during receptor stimulation and can be adapted to other research questions related to this topic.

EB1 binding restricts STIM1 translocation to ER-PM junctions and regulates store-operated Ca2+ entry.

The endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) after ER Ca2+ depletion. STIM1 also interacts with EB1 and dynamically tracks microtubule (MT) plus ends. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with a synthetic construct approach, we found that EB1 binding constitutes a trapping mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. By trapping STIM1 molecules at dynamic contacts between the ER and MT plus ends, EB1 binding delayed STIM1 translocation to ER-PM junctions during ER Ca2+ depletion and prevented excess SOCE and ER Ca2+ overload. Our study suggests that STIM1-EB1 interaction shapes the kinetics and amplitude of local SOCE in cellular regions with growing MTs and contributes to spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.

Cortical actin contributes to spatial organization of ER-PM junctions.

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate crucial activities ranging from Ca2+ signaling to lipid metabolism. Spatial organization of ER-PM junctions may modulate the extent and location of these cellular activities. However, the morphology and distribution of ER-PM junctions are not well characterized. Using photoactivated localization microscopy, we reveal that the contact area of single ER-PM junctions is mainly oblong with the dimensions of ∼120 nm × âˆ¼80 nm in HeLa cells. Using total internal reflection fluorescence microscopy and structure illumination microscopy, we show that cortical actin contributes to spatial distribution and stability of ER-PM junctions. Further functional assays suggest that intact F-actin architecture is required for phosphatidylinositol 4,5-bisphosphate homeostasis mediated by Nir2 at ER-PM junctions. Together, our study provides quantitative information on spatial organization of ER-PM junctions that is in part regulated by F-actin. We envision that functions of ER-PM junctions can be differentially regulated through dynamic actin remodeling during cellular processes.

RASSF4 controls SOCE and ER-PM junctions through regulation of PI(4,5)P2.

RAS association domain family 4 (RASSF4) is involved in tumorigenesis and regulation of the Hippo pathway. In this study, we identify new functional roles of RASSF4. First, we discovered that RASSF4 regulates store-operated Ca2+ entry (SOCE), a fundamental Ca2+ signaling mechanism, by affecting the translocation of the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) to ER-plasma membrane (PM) junctions. It was further revealed that RASSF4 regulates the formation of ER-PM junctions and the ER-PM tethering function of extended synaptotagmins E-Syt2 and E-Syt3. Moreover, steady-state PM phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) levels, important for localization of STIM1 and E-Syts at ER-PM junctions, were reduced in RASSF4-knockdown cells. Furthermore, we demonstrated that RASSF4 interacts with and regulates the activity of adenosine diphosphate ribosylation factor 6 (ARF6), a small G protein and upstream regulator of type I phosphatidylinositol phosphate kinases (PIP5Ks) and PM PI(4,5)P2 levels. Overall, our study suggests that RASSF4 controls SOCE and ER-PM junctions through ARF6-dependent regulation of PM PI(4,5)P2 levels, pivotal for a variety of physiological processes.

ER-plasma membrane junctions: Why and how do we study them?

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are membrane microdomains important for communication between the ER and the PM. ER-PM junctions were first reported in muscle cells in 1957, but mostly ignored in non-excitable cells due to their scarcity and lack of functional significance. In 2005, the discovery of stromal interaction molecule 1 (STIM1) mediating a universal Ca2+ feedback mechanism at ER-PM junctions in mammalian cells led to a resurgence of research interests toward ER-PM junctions. In the past decade, several major advancements have been made in this emerging topic in cell biology, including the generation of tools for labeling ER-PM junctions and the unraveling of mechanisms underlying regulation and functions of ER-PM junctions. This review summarizes early studies, recently developed tools, and current advances in the characterization and understanding of ER-PM junctions. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

Homeostatic regulation of the PI(4,5)P2-Ca(2+) signaling system at ER-PM junctions.

The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-Ca(2+) signaling system is important for cell activation in response to various extracellular stimuli. This signaling system is initiated by receptor-induced hydrolysis of PI(4,5)P2 in the plasma membrane (PM) to generate the soluble second messenger inositol 1,4,5-trisphosphate (IP3). IP3 subsequently triggers the release of Ca(2+) from the endoplasmic reticulum (ER) store to the cytosol to activate Ca(2+)-mediated responses, such as secretion and proliferation. The consumed PM PI(4,5)P2 and ER Ca(2+) must be quickly restored to sustain signaling responses, and to maintain the homeostasis of PI(4,5)P2 and Ca(2+). Since phosphatidylinositol (PI), the precursor lipid for PM PI(4,5)P2, is synthesized in the ER membrane, and a Ca(2+) influx across the PM is required to refill the ER Ca(2+) store, efficient communications between the ER and the PM are critical for the homeostatic regulation of the PI(4,5)P2-Ca(2+) signaling system. This review describes the major findings that established the framework of the PI(4,5)P2-Ca(2+) signaling system, and recent discoveries on feedback control mechanisms at ER-PM junctions that sustain the PI(4,5)P2-Ca(2+) signaling system. Particular emphasis is placed on the characterization of ER-PM junctions where efficient communications between the ER and the PM occur, and the activation mechanisms of proteins that dynamically localize to ER-PM junctions to provide the feedback control during PI(4,5)P2-Ca(2+) signaling, including the ER Ca(2+) sensor STIM1, the extended synaptotagmin E-Syt1, and the PI transfer protein Nir2. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions.

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) at the plasma membrane (PM) constitutively controls many cellular functions, and its hydrolysis via receptor stimulation governs cell signaling. The PI transfer protein Nir2 is essential for replenishing PM PIP2 following receptor-induced hydrolysis, but key mechanistic aspects of this process remain elusive. Here, we demonstrate that PI at the membrane of the endoplasmic reticulum (ER) is required for the rapid replenishment of PM PIP2 mediated by Nir2. Nir2 detects PIP2 hydrolysis and translocates to ER-PM junctions via binding to phosphatidic acid. With distinct phosphatidic acid binding abilities and PI transfer protein activities, Nir2 and its homolog Nir3 differentially regulate PIP2 homeostasis in cells during intense receptor stimulation and in the resting state, respectively. Our study reveals that Nir2 and Nir3 work in tandem to achieve different levels of feedback based on the consumption of PM PIP2 and function at ER-PM junctions to mediate nonvesicular lipid transport between the ER and the PM.

A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.

Functional micropeptides can be concealed within RNAs that appear to be noncoding. We discovered a conserved micropeptide, which we named myoregulin (MLN), encoded by a skeletal muscle-specific RNA annotated as a putative long noncoding RNA. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), which inhibit SERCA, the membrane pump that controls muscle relaxation by regulating Ca(2+) uptake into the sarcoplasmic reticulum (SR). MLN interacts directly with SERCA and impedes Ca(2+) uptake into the SR. In contrast to PLN and SLN, which are expressed in cardiac and slow skeletal muscle in mice, MLN is robustly expressed in all skeletal muscle. Genetic deletion of MLN in mice enhances Ca(2+) handling in skeletal muscle and improves exercise performance. These findings identify MLN as an important regulator of skeletal muscle physiology and highlight the possibility that additional micropeptides are encoded in the many RNAs currently annotated as noncoding.

Effect of gonadotropin-releasing hormone agonist on ES-2 ovarian cancer cells.

OBJECTIVE: Gonadotropin-releasing hormone (GnRH) receptor is found in the ovarian tissue, including epithelial ovarian cancer (EOC), suggesting that GnRH agonists may have direct action on EOC. MATERIALS AND METHODS: Ovarian clear cell cancer (ES-2) cells were treated with low-dose GnRH agonist with/without low-dose paclitaxel (1 µM D-Lys(6) with/without 0.5 µM or 1.0 µM paclitaxel). Growth and behavior of ES-2 cells were evaluated. RESULTS: Use of either D-Lys(6) or paclitaxel or a combination of the two did not affect the morphology and growth pattern of ES-2 cells. However, ability of migration and invasion of ES-2 cells was significantly decreased in either use of D-Lys(6) or paclitaxel and more apparent with the combination. Type I GnRH receptor expression of ES-2 was not altered significantly by the combination. CONCLUSION: GnRH agonist might modify the ES-2 ovarian cancer cells, and its role might be independent, additional or synergistic, suggesting the potential role of the use of GnRH agonist in the management of clear cell type of the ovarian cancer. However, the results of this study were derived using ES-2 ovarian cancer cells, and might not be valid in other cell types of ovarian cancers.