Mechanisms Of Presynaptic CaV2 Calcium Channel Localization In Caenorhabditis Elegans

Neurotransmitter release at nerve terminals is a fundamental aspect of communication in the nervous system. Voltage changes in the presynaptic membrane are sensed by presynaptic voltage-gated calcium channels that mediate calcium influx at the nerve terminals to execute exocytosis of various kinds of neurotransmitters. Defects in presynaptic calcium channels lead to many neurological disorders, emphasizing the importance of these channels in the regulation of neuronal activities in the brain. Previous physiological studies have focused on opening kinetics of the channels and their modulation by auxiliary subunits. Cell biological questions such as trafficking and clustering of the channels at the presynaptic site, however, have remained largely unanswered, partly due to a lack of an in vivo assay system to monitor calcium channel biogenesis in intact animals. In my thesis, I established an in vivo system to visualize the UNC-2 alpha1 subunit of the C. elegans CaV2 channel. I showed that GFP-tagged UNC-2 is localized to presynaptic active zones of sensory and motor neurons. Using this system, I conducted a direct visual genetic screen for mutants that are defective in UNC-2 localization, identifying three genes, calf-1, unc-36 and pqn-53/calf-2. CALF-1 is a neuronal-specific one-pass transmembrane protein that resides in the endoplasmic reticulum, and is required for endoplasmic reticulum exit of UNC-2. Structure-function analysis revealed that the transmembrane domain and the cytosolic arginine-based basic motifs are important for the function of CALF-1. Acute induction of calf-1 mobilizes preexisting UNC-2 for delivery to synapses, consistent with a direct trafficking role. The calcium channel alpha2-delta auxiliary subunit UNC-36 is also required for endoplasmic reticulum exit of UNC-2, but has additional functions. The polyglutamine protein pqn-53 is localized to the nucleus; PQN-53 inhibits a non-canonical unfolded protein response pathway, and activation of this pathway in pqn-53 mutants leads to the reduced expression of UNC-2 and other transmembrane proteins. Furthermore, pqn-53 mutants are resistant to killing by pathogenic bacteria, potentially due to constitutive activation of the non-canonical unfolded protein response. pqn-53 couples multipass transmembrane protein biogenesis with endoplasmic reticulum stress, and provides insights into the regulation of pathogen resistance by the endoplasmic reticulum stress-sensing pathway

Meeting Report from the 2019 "Organelle Zone" Symposium in Osaka, Japan

International audienceOn May 29 at the Osaka University Hospital, Japan, the "Organelle Zones" research grant group (see http://organellezone.org/english/) organized a one day symposium for its own members and four guest speakers, with about 60 attendees. The research group studies three different ways in which regions within organelles carry out functions distinct from other parts of the organelle. Work at this sub-organellar level is increasingly recognised as an important aspect of cell biology. The group's projects are divided into these themes with 9 Principal Investigators and 18 Co-Investigators over 5 years. The symposium, followed a similar meeting in 2018, and had 4 external speakers and 4 internal members of the consortium. The talks were divided into three sessions, each show-casing one way of sub-compartmentalising organelles into zones

Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in C. elegans

Mutations in pre-synaptic voltage gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(zf35gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(zf35gf) mutants. unc-2(zf35gf) mutants display increased cholinergic-and decreased GABAergic-transmission. Moreover, increased cholinergic transmission in unc-2(zf35gf) mutants leads to an increase of cholinergic synapses and a TAX-6/calcineurin dependent reduction of GABA synapses. Our studies reveal mechanisms through which CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system.Fil: Huang, Yung Chi. University of Massachussets; Estados UnidosFil: Pirri, Jennifer K.. University of Massachussets; Estados UnidosFil: Rayes, Diego Hernán. University of Massachussets; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Gao, Shangbang. Mount Sinai Hospital; Estados UnidosFil: Mulcahy, Ben. Mount Sinai Hospital; Estados UnidosFil: Grant, Jeff. University of Massachussets; Estados UnidosFil: Saheki, Yasunori. The Rockefeller University; Estados UnidosFil: Francis, Michael M.. University of Massachussets; Estados UnidosFil: Zhen, Mei. University of Toronto; Canadá. Mount Sinai Hospital; Estados UnidosFil: Alkema, Mark J.. University of Massachussets; Estados Unido

Left-right olfactory asymmetry results from antagonistic functions of voltage-activated calcium channels and the Raw repeat protein OLRN-1 in C. elegans

<p>Abstract</p> <p>Background</p> <p>The left and right AWC olfactory neurons in <it>Caenorhabditis elegans </it>differ in their functions and in their expression of chemosensory receptor genes; in each animal, one AWC randomly takes on one identity, designated AWC^OFF, and the contralateral AWC becomes AWC^ON. Signaling between AWC neurons induces left-right asymmetry through a gap junction network and a claudin-related protein, which inhibit a calcium-regulated MAP kinase pathway in the neuron that becomes AWC^ON.</p> <p>Results</p> <p>We show here that the asymmetry gene <it>olrn-1 </it>acts downstream of the gap junction and claudin genes to inhibit the calcium-MAP kinase pathway in AWC^ON. OLRN-1, a protein with potential membrane-association domains, is related to the <it>Drosophila </it>Raw protein, a negative regulator of JNK mitogen-activated protein (MAP) kinase signaling. <it>olrn-1 </it>opposes the action of two voltage-activated calcium channel homologs, <it>unc-2 </it>(CaV2) and <it>egl-19 </it>(CaV1), which act together to stimulate the calcium/calmodulin-dependent kinase CaMKII and the MAP kinase pathway. Calcium channel activity is essential in AWC^OFF, and the two AWC neurons coordinate left-right asymmetry using signals from the calcium channels and signals from <it>olrn-1</it>.</p> <p>Conclusion</p> <p><it>olrn-1 </it>and voltage-activated calcium channels are mediators and targets of AWC signaling that act at the transition between a multicellular signaling network and cell-autonomous execution of the decision. We suggest that the asymmetry decision in AWC results from the intercellular coupling of voltage-regulated channels, whose cross-regulation generates distinct calcium signals in the left and right AWC neurons. The interpretation of these signals by the kinase cascade initiates the sustained difference between the two cells.</p

SMP domain proteins in membrane lipid dynamics

Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved family of proteins in eukaryotes that localize between the endoplasmic reticulum (ER) and either the plasma membrane (PM) or other organelles. They are involved in tethering of these membrane contact sites through interaction with other proteins and membrane lipids. Recent structural and biochemical studies have demonstrated that SMP domain proteins transport a wide variety of lipid species by the ability of the SMP domain to harbor lipids through its unique hydrophobic cavity. Growing evidence suggests that SMP domain proteins play critical roles in cell physiology by their actions at membrane contact sites. In this review, we summarize the functions of SMP domain proteins and their direct roles in lipid transport across different membrane compartments. We also discuss their physiological functions in organisms as well as “bypass” pathways that act in parallel with SMP domain proteins at membrane contact sites.Ministry of Education (MOE)Accepted versionWe apologize to all the investigators whose work could not be cited due to space limitations. We thank Bilge Ercan for her help in the visualization of the structures of the SMP domains and Jingbo Sun, Nur Raihanah Binte Mohd Harion, Tomoki Naito, and Dylan Hong Zheng Koh for their constructive feedback to the manuscript. Work from the authors related to this review has been supported in part by a Grant-in-Aid for Young Scientists (A) from the Japan Society for the Promotion of Science (17H05065), the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2017-T2-2-001), Academic Research Fund Tier 1 (MOE2018-T1-001-023), a Nanyang Assistant Professorship (NAP), and a Lee Kong Chian School of Medicine startup grant (LKCMedicine-SUG) to Y.S

GRAMD1-mediated accessible cholesterol sensing and transport

Cholesterol, an essential lipid for cell signaling and structural integrity of cellular membranes, is highly enriched in the plasma membrane (PM). However, the regulatory mechanisms that control its biosynthesis and uptake both reside in the endoplasmic reticulum (ER). Thus, the ER needs to constantly monitor the levels of PM cholesterol. This is in part mediated by regulated transport of a biochemically defined pool of cholesterol, termed "accessible" cholesterol, from the PM to the ER via evolutionarily conserved ER-anchored lipid transfer proteins, the GRAMD1s/Asters (GRAMD1a/1b/1c) (Lam/Ltc proteins in yeast). GRAMD1s possess cytosolically exposed GRAM domain and StART-like domain followed by a transmembrane ER anchor. They form homo- and hetero-meric complexes and move to the contacts formed between the ER and the PM by sensing a transient expansion of the accessible pool of cholesterol in the PM via the GRAM domain and facilitate its extraction and transport to the ER via the StART-like domain. The GRAMD1b GRAM domain possesses distinct, but synergistic sites, for recognizing accessible cholesterol and anionic lipids, including phosphatidylserine, within the PM. This property of the GRAM domain contributes to regulated tethering of the PM to ER membrane where GRAMD1s are anchored and fine-tunes StART-like domain-dependent accessible cholesterol transport. Thus, cells use GRAMD1s to sense the levels of cholesterol in the PM and regulate transport of accessible PM cholesterol to the ER in order to maintain cholesterol homeostasis.Ministry of Education (MOE)This work was supported in part by the Ministry of Education, Singapore, under its Academic Research Fund Tier 2 Award (MOE2017-T2-2-001), a Nanyang Assistant Professorship (NAP), and a Lee Kong Chian School of Medicine startup grant (LKCMedicine-SUG) to Y.S. T.N. was supported by a fellowship from the Japanese Society for Promotion of Science

Endoplasmic reticulum–plasma membrane contact sites

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER–PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins

The Extended-Synaptotagmins

The extended-synaptotagmins (tricalbins in yeast) derive their name from their partial domain structure similarity to the synaptotagmins, which are characterized by an N-terminal membrane anchor and cytosolically exposed C2 domains. However, they differ from the synaptotagmins in localization and function. The synaptotagmins tether secretory vesicles, including synaptic vesicles, to the plasma membrane (PM) via their C2 domains and regulate their Ca2+ triggered exocytosis. In contrast, the extended-synaptotagmins are resident proteins of the endoplasmic reticulum (ER), which tether this organelle to the plasma membrane via their C2 domains, but not as a premise to fusion of the two membranes. They transport glycerolipids between the two bilayers via their lipid-harboring SMP domains and Ca2+ regulates their membrane tethering and lipid transport function. Additionally, the extended-synaptotagmins are more widely expressed in different organisms, as they are present not only in animal cells, but also in fungi and plants, which do not express the synaptotagmins. Thus, they have a more general function than the synaptotagmins, whose appearance in animal species correlated with the occurrence of Ca2+ triggered exocytosis. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.Accepted versio

Ca 2+ releases E‐Syt1 autoinhibition to couple ER‐plasma membrane tethering with lipid transport

The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E-Syt1 tethers and transports lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables lipid transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.Accepted versio

INPP5K and Atlastin-1 maintain the nonuniform distribution of ER-plasma membrane contacts in neurons

In neurons, the ER extends throughout all cellular processes, forming multiple contacts with the plasma membrane (PM) to fine-tune neuronal physiology. However, the mechanisms that regulate the distribution of neuronal ER-PM contacts are not known. Here, we used the Caenorhabditis elegans DA9 motor neuron as our model system and found that neuronal ER-PM contacts are enriched in soma and dendrite and mostly absent in axons. Using forward genetic screen, we identified that the inositol 5-phosphatase, CIL-1 (human INPP5K), and the dynamin-like GTPase, ATLN-1 (human Atlastin-1), help to maintain the non-uniform, somatodendritic enrichment of neuronal ER-PM contacts. Mechanistically, CIL-1 acts upstream of ATLN-1 to maintain the balance between ER tubules and sheets. In mutants of CIL-1 or ATLN-1, ER sheets expand and invade into the axon. This is accompanied by the ectopic formation of axonal ER-PM contacts and defects in axon regeneration following laser-induced axotomy. As INPP5K and Atlastin-1 have been linked to neurological disorders, the unique distribution of neuronal ER-PM contacts maintained by these proteins may support neuronal resilience during the onset and progression of these diseases.Ministry of Education (MOE)Nanyang Technological UniversityPublished versionThis work was supported in part by the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2017-T2-2-001), a Nanyang Assistant Professorship, a Lee Kong Chian School of Medicine startup grant (LKCMedicine-SUG), and an Ageing Research Institute for Society and Education (ARISE) seed grant (ARISE/2017/7) to Y Saheki