Kalium channelrhodopsins effectively inhibit neurons.

The analysis of neural circuits has been revolutionized by optogenetic methods. Light-gated chloride-conducting anion channelrhodopsins (ACRs)-recently emerged as powerful neuron inhibitors. For cells or sub-neuronal compartments with high intracellular chloride concentrations, however, a chloride conductance can have instead an activating effect. The recently discovered light-gated, potassium-conducting, kalium channelrhodopsins (KCRs) might serve as an alternative in these situations, with potentially broad application. As yet, KCRs have not been shown to confer potent inhibitory effects in small genetically tractable animals. Here, we evaluated the utility of KCRs to suppress behavior and inhibit neural activity in Drosophila, Caenorhabditis elegans, and zebrafish. In direct comparisons with ACR1, a KCR1 variant with enhanced plasma-membrane trafficking displayed comparable potency, but with improved properties that include reduced toxicity and superior efficacy in putative high-chloride cells. This comparative analysis of behavioral inhibition between chloride- and potassium-selective silencing tools establishes KCRs as next-generation optogenetic inhibitors for in vivo circuit analysis in behaving animals.

Visualization of accessible cholesterol using a GRAM domain-based biosensor.

Cholesterol is important for membrane integrity and cell signaling, and dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including neurodegenerative disorders. While regulated transport of a specific pool of cholesterol, known as "accessible cholesterol", contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to monitor accessible cholesterol in live cells remain limited. Here, we engineer a highly sensitive accessible cholesterol biosensor by taking advantage of the cholesterol-sensing element (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b. Using this cholesterol biosensor, which we call GRAM-W, we successfully visualize in real time the distribution of accessible cholesterol in many different cell types, including human keratinocytes and iPSC-derived neurons, and show differential dependencies on cholesterol biosynthesis and uptake for maintaining levels of accessible cholesterol. Furthermore, we combine GRAM-W with a dimerization-dependent fluorescent protein (ddFP) and establish a strategy for the ultrasensitive detection of accessible plasma membrane cholesterol. These tools will allow us to obtain important insights into the molecular mechanisms by which the distribution of cellular cholesterol is regulated.

Regulation of cellular cholesterol distribution via non-vesicular lipid transport at ER-Golgi contact sites.

Abnormal distribution of cellular cholesterol is associated with numerous diseases, including cardiovascular and neurodegenerative diseases. Regulated transport of cholesterol is critical for maintaining its proper distribution in the cell, yet the underlying mechanisms remain unclear. Here, we show that lipid transfer proteins, namely ORP9, OSBP, and GRAMD1s/Asters (GRAMD1a/GRAMD1b/GRAMD1c), control non-vesicular cholesterol transport at points of contact between the ER and the trans-Golgi network (TGN), thereby maintaining cellular cholesterol distribution. ORP9 localizes to the TGN via interaction between its tandem α-helices and ORP10/ORP11. ORP9 extracts PI4P from the TGN to prevent its overaccumulation and suppresses OSBP-mediated PI4P-driven cholesterol transport to the Golgi. By contrast, GRAMD1s transport excess cholesterol from the Golgi to the ER, thereby preventing its build-up. Cells lacking ORP9 exhibit accumulation of cholesterol at the Golgi, which is further enhanced by additional depletion of GRAMD1s with major accumulation in the plasma membrane. This is accompanied by chronic activation of the SREBP-2 signalling pathway. Our findings reveal the importance of regulated lipid transport at ER-Golgi contacts for maintaining cellular cholesterol distribution and homeostasis.

PDZD-8 and TEX-2 regulate endosomal PI(4,5)P2 homeostasis via lipid transport to promote embryogenesis in C. elegans.

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.

Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes.

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.

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.

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.

Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b.

Cholesterol is essential for cell physiology. Transport of the "accessible" pool of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER) by ER-localized GRAMD1 proteins (GRAMD1a/1b/1c) contributes to cholesterol homeostasis. However, how cells detect accessible cholesterol within the PM remains unclear. We show that the GRAM domain of GRAMD1b, a coincidence detector for anionic lipids, including phosphatidylserine (PS), and cholesterol, possesses distinct but synergistic sites for sensing accessible cholesterol and anionic lipids. We find that a mutation within the GRAM domain of GRAMD1b that is associated with intellectual disability in humans specifically impairs cholesterol sensing. In addition, we identified another point mutation within this domain that enhances cholesterol sensitivity without altering its PS sensitivity. Cell-free reconstitution and cell-based assays revealed that the ability of the GRAM domain to sense accessible cholesterol regulates membrane tethering and determines the rate of cholesterol transport by GRAMD1b. Thus, cells detect the codistribution of accessible cholesterol and anionic lipids in the PM and fine-tune the non-vesicular transport of PM cholesterol to the ER via GRAMD1s.

Regulation of Plasma Membrane Sterol Homeostasis by Nonvesicular Lipid Transport.

Sterol contributes to the structural integrity of cellular membranes and plays an important role in the regulation of cell signaling in eukaryotes. It is either produced in the endoplasmic reticulum or taken up from the extracellular environment. In most eukaryotic cells, however, the majority of sterol is enriched in the plasma membrane. Thus, the transport of sterol between the plasma membrane and other organelles, including the endoplasmic reticulum, is crucial for maintaining sterol homeostasis. While vesicular transport that relies on membrane budding and fusion reactions plays an important role in bulk sterol transport, this mode of transport is slow and non-selective. Growing evidence suggests a critical role of nonvesicular transport mediated by evolutionarily conserved families of lipid transfer proteins in more rapid and selective delivery of sterol. Some lipid transfer proteins act primarily at the sites of contacts formed between the endoplasmic reticulum and other organelles or the plasma membrane without membrane fusion. In this review, we describe the similarities and differences of sterol biosynthesis and uptake in mammals and yeast and discuss the role of their lipid transfer proteins in maintaining plasma membrane sterol homeostasis.

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.

Movement of accessible plasma membrane cholesterol by the GRAMD1 lipid transfer protein complex.

Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholesterol to the ER in order to counteract an acute increase of PM cholesterol, thereby activating non-vesicular cholesterol transport.

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

On 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 Caenorhabditis 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.

Ca2+ 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.

Endoplasmic Reticulum - Plasma Membrane Crosstalk Mediated by the Extended Synaptotagmins.

The endoplasmic reticulum (ER) possesses multiplicity of functions including protein synthesis, membrane lipid biogenesis, and Ca2+ storage and has broad localization throughout the cell. While the ER and most other membranous organelles are highly interconnected via vesicular traffic that relies on membrane budding and fusion reactions, the ER forms direct contacts with virtually all other membranous organelles, including the plasma membrane (PM), without membrane fusion. Growing evidence suggests that these contacts play major roles in cellular physiology, including the regulation of Ca2+ homeostasis and signaling and control of cellular lipid homeostasis. Extended synaptotagmins (E-Syts) are evolutionarily conserved family of ER-anchored proteins that tether the ER to the PM in PM PI(4,5)P2-dependent and cytosolic Ca2+-regulated manner. In addition, E-Syts possess a cytosolically exposed lipid-harboring module that confers the ability to transfer/exchange glycerolipids between the ER and the PM at E-Syts-mediated ER-PM contacts. In this chapter, the functions of ER-PM contacts and their role in non-vesicular lipid transport with special emphasis on the crosstalk between the two bilayers mediated by E-Syts will be discussed.

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.

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.

Endosome-ER Contacts Control Actin Nucleation and Retromer Function through VAP-Dependent Regulation of PI4P.

VAP (VAPA and VAPB) is an evolutionarily conserved endoplasmic reticulum (ER)-anchored protein that helps generate tethers between the ER and other membranes through which lipids are exchanged across adjacent bilayers. Here, we report that by regulating PI4P levels on endosomes, VAP affects WASH-dependent actin nucleation on these organelles and the function of the retromer, a protein coat responsible for endosome-to-Golgi traffic. VAP is recruited to retromer budding sites on endosomes via an interaction with the retromer SNX2 subunit. Cells lacking VAP accumulate high levels of PI4P, actin comets, and trans-Golgi proteins on endosomes. Such defects are mimicked by downregulation of OSBP, a VAP interactor and PI4P transporter that participates in VAP-dependent ER-endosomes tethers. These results reveal a role of PI4P in retromer-/WASH-dependent budding from endosomes. Collectively, our data show how the ER can control budding dynamics and association with the cytoskeleton of another membrane by direct contacts leading to bilayer lipid modifications.

Control of plasma membrane lipid homeostasis by the extended synaptotagmins.

Acute metabolic changes in plasma membrane (PM) lipids, such as those mediating signalling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the extended synaptotagmins (E-Syts), endoplasmic reticulum (ER) proteins that function as PtdIns(4,5)P2- and Ca(2+)-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro, and this transfer requires Ca(2+) and their lipid-harbouring SMP domain. Genome-edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but exhibit enhanced and sustained accumulation of PM diacylglycerol following PtdIns(4,5)P2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syt-dependent ER-PM tethers in response to stimuli that cleave PtdIns(4,5)P2 and elevate Ca(2+) may help reverse accumulation of diacylglycerol in the PM by transferring it to the ER for metabolic recycling.