scholarly journals Extended Synaptotagmin is a presynaptic ER Ca2+ sensor that promotes neurotransmission and synaptic growth in Drosophila

2017 ◽  
Author(s):  
Koto Kikuma ◽  
Daniel Kim ◽  
David Sutter ◽  
Xiling Li ◽  
Dion K. Dickman
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Synaptic Activity ◽  
Synaptic Growth ◽  
Synaptic Physiology ◽  
Lipid Exchange ◽  
Membrane Tethering ◽  
Conserved Gene ◽  
Neuronal Functions

ABSTRACTThe endoplasmic reticulum (ER) is an extensive presynaptic organelle, exerting important influences at synapses by responding to Ca2+ and modulating transmission, growth, lipid metabolism, and membrane trafficking. Despite intriguing evidence for these crucial functions, how presynaptic ER influences synaptic physiology remains enigmatic. To gain insight into this question, we have generated and characterized mutations in the single Extended Synaptotagmin (Esyt) ortholog in Drosophila. Esyts are evolutionarily conserved ER proteins with Ca2+ sensing domains that have recently been shown to orchestrate membrane tethering and lipid exchange between the ER and plasma membrane. We first demonstrate that Esyt localizes to an extensive ER structure that invades presynaptic terminals at the neuromuscular junction. Next, we show that synaptic growth, structure, function, and plasticity are surprisingly unperturbed at synapses lacking Esyt expression. However, presynaptic overexpression of Esyt leads to enhanced synaptic growth, neurotransmission, and sustainment of the vesicle pool during intense levels of activity, suggesting that elevated Esyt at the ER promotes constitutive membrane trafficking or lipid exchange with the plasma membrane. Finally, we find that Esyt mutants fail to maintain basal neurotransmission and short term plasticity at elevated extracellular Ca2+, consistent with Esyt functioning as an ER Ca2+ sensor that modulates synaptic activity. Thus, we identify Esyt as a presynaptic ER Ca2+ sensor that can promote neurotransmission and synaptic growth, revealing the first in vivo neuronal functions of this conserved gene family.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals The endoplasmic reticulum-plasma membrane tethering protein TMEM24 is a regulator of cellular Ca2+ homeostasis

2021 ◽  
Author(s):  
Beichen Xie ◽  
Styliani Panagiotou ◽  
Jing Cen ◽  
Patrick Gilon ◽  
Peter Bergsten ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Insulin Secretion ◽  
Lipid Transfer Protein ◽  
Underlying Mechanism ◽  
Lipid Transfer ◽  
Β Cells ◽  
Transfer Protein ◽  
Lipid Exchange ◽  
Membrane Tethering

Endoplasmic reticulum (ER) - plasma membrane (PM) contacts are sites of lipid exchange and Ca2+ transport, and both lipid transport proteins and Ca2+ channels specifically accumulate at these locations. In pancreatic β-cells, both lipid- and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24 dynamically localize to ER-PM contact sites and provide phosphatidylinositol, a precursor of PI(4)P and PI(4,5)P2, to the plasma membrane. β-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion but the underlying mechanism is not known. We now show that TMEM24 only weakly interact with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Release of TMEM24 into the bulk ER membrane also enables direct interactions with mitochondria, and we report that loss of TMEM24 results in excessive accumulation of Ca2+ in both the ER and mitochondria and in impaired mitochondria function.

Impulse activity of a crayfish motoneuron regulated its neuromuscular synaptic properties

1989 ◽  
Vol 61 (1) ◽  
pp. 91-96 ◽  
Author(s):  
G. A. Lnenicka ◽  
H. L. Atwood
Keyword(s):  
Muscle Activity ◽  
Transmitter Release ◽  
Impulse Activity ◽  
Peripheral Region ◽  
Synaptic Activity ◽  
Synaptic Physiology ◽  
Neuromuscular Synapses ◽  
Long Term Changes

1. Previous studies have demonstrated that initial transmitter release, fatigability, and the morphology of identified crayfish neuromuscular synapses adapt to long-term changes in motoneuron impulse activity. 2. Experiments were performed to determine whether these long-term, adaptive alterations in neuromuscular synaptic physiology are triggered by changes in neuromuscular synaptic activity, muscle activity, or neuronal impulse activity. The fast closer excitor of the crayfish claw, a phasic motoneuron, was studied. Either the central or the peripheral region of the motoneuron was selectively stimulated in vivo by blocking impulse activity midway along the motor axon with localized application of tetrodotoxin and stimulating either central or distal to the blocked region. 3. Neither muscle activity nor transmitter release from the neuromuscular synapses was required to trigger the changes in synaptic physiology. Stimulation central to the block induced changes in neuromuscular transmission that included a long-lasting decrease in initial transmitter release and increased fatique resistance. 4. Because peripheral stimulation also produced decreased initial transmitter release, it appears that increased impulse activity in either region of the motoneuron can produce the synaptic changes. These results along with earlier findings suggest that neuronal depolarization induces adaptive, long-term changes in synapses. 5. These results are discussed in relation to findings at vertebrate and invertebrate synapses.

scholarly journals Apical targeting of syntaxin 3 is essential for epithelial cell polarity

2006 ◽  
Vol 173 (6) ◽  
pp. 937-948 ◽  
Author(s):  
Nikunj Sharma ◽  
Seng Hui Low ◽  
Saurav Misra ◽  
Bhattaram Pallavi ◽  
Thomas Weimbs
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Membrane Trafficking ◽  
Cell Polarization ◽  
Apical Plasma Membrane ◽  
Membrane Expression ◽  
Tight Junction Formation ◽  
Necessary And Sufficient ◽  
Syntaxin 3

In polarized epithelial cells, syntaxin 3 localizes to the apical plasma membrane and is involved in membrane fusion of apical trafficking pathways. We show that syntaxin 3 contains a necessary and sufficient apical targeting signal centered around a conserved FMDE motif. Mutation of any of three critical residues within this motif leads to loss of specific apical targeting. Modeling based on the known structure of syntaxin 1 revealed that these residues are exposed on the surface of a three-helix bundle. Syntaxin 3 targeting does not require binding to Munc18b. Instead, syntaxin 3 recruits Munc18b to the plasma membrane. Expression of mislocalized mutant syntaxin 3 in Madin-Darby canine kidney cells leads to basolateral mistargeting of apical membrane proteins, disturbance of tight junction formation, and loss of ability to form an organized polarized epithelium. These results indicate that SNARE proteins contribute to the overall specificity of membrane trafficking in vivo, and that the polarity of syntaxin 3 is essential for epithelial cell polarization.

scholarly journals EHD Proteins Associate with Syndapin I and II and Such Interactions Play a Crucial Role in Endosomal Recycling

2005 ◽  
Vol 16 (8) ◽  
pp. 3642-3658 ◽  
Author(s):  
Anne Braun ◽  
Roser Pinyol ◽  
Regina Dahlhaus ◽  
Dennis Koch ◽  
Paul Fonarev ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Crucial Role ◽  
Mutational Analysis ◽  
Protein Complexes ◽  
Binding Interface ◽  
Endosomal Recycling ◽  
Eh Domain ◽  
Ehd Proteins

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin–EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.

scholarly journals The endoplasmic reticulum-plasma membrane tethering protein TMEM24 is a regulator of cellular Ca2+ homeostasis

2021 ◽  
Author(s):  
Beichen Xie ◽  
Styliani Panagiotou ◽  
Jing Cen ◽  
Patrick Gilon ◽  
Peter Bergsten ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Insulin Secretion ◽  
Lipid Transfer Protein ◽  
Underlying Mechanism ◽  
Lipid Transfer ◽  
Β Cells ◽  
Atp Production ◽  
Lipid Exchange ◽  
Membrane Tethering

Endoplasmic reticulum (ER) - plasma membrane (PM) contacts are sites of lipid exchange and Ca2+ transport, and both lipid transport proteins and Ca2+ channels specifically accumulate at these locations. In pancreatic β-cells, both lipid- and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24 dynamically localize to ER-PM contact sites and provide phosphatidylinositol, a precursor of PI(4)P and PI(4,5)P2, to the plasma membrane. β-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion but the underlying mechanism is not known. We now show that TMEM24 only weakly interact with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Loss of TMEM24 results in hyper-accumulation of Ca2+ in the ER and in excess Ca2+ entry into mitochondria, with resulting impairment in glucose-stimulated ATP production.

scholarly journals Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 124
Author(s):  
Ilkka Fagerlund ◽  
Antonios Dougalis ◽  
Anastasia Shakirzyanova ◽  
Mireia Gómez-Budia ◽  
Anssi Pelkonen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Synaptic Activity ◽  
Repetitive Firing ◽  
Electrophysiological Recordings ◽  
Induced Pluripotent ◽  
Neuronal Functions

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.

scholarly journals Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

2011 ◽  
Vol 137 (2) ◽  
pp. 137-154 ◽  
Author(s):  
Michael Fine ◽  
Marc C. Llaguno ◽  
Vincenzo Lariccia ◽  
Mei-Jung Lin ◽  
Alp Yaradanakul ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Atp Hydrolysis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Hek293 Cells ◽  
Nitric Oxide Donor ◽  
Cytoskeleton Remodeling ◽  
Nonionic Detergents

The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4–64, into vesicles and loss of reversible FM 4–64 binding confirm that 40–75% of the cell surface is internalized. Ongoing MEND stops in 2–4 s when amphipaths are removed, and amphipaths are without effect from the cytoplasmic side. Thus, expansion of the outer monolayer is critical. As found for Ca-activated MEND, vesicles formed are <100 nm in diameter, membrane ruffles are lost, and β-cyclodextrin treatments are inhibitory. However, amphipath-activated MEND does not require Ca transients, adenosine triphosphate (ATP) hydrolysis, G protein cycling, dynamins, or actin cytoskeleton remodeling. With elevated cytoplasmic ATP (>5 mM), MEND can reverse completely and be repeated multiple times in BHK and HEK293 cells, but not cardiac myocytes. Reversal is blocked by N-ethylmaleimide and a nitric oxide donor, nitroprusside. Constitutively expressed Na/Ca exchangers internalize roughly in proportion to surface membrane, whereas Na/K pump activities decrease over-proportionally. Sodium dodecyl sulfate and dodecylglucoside do not cause MEND during their application, but MEND occurs rapidly when they are removed. As monitored capacitively, the binding of these detergents decreases with MEND, whereas TX100 binding does not decrease. In summary, nonionic detergents can fractionate the plasma membrane in vivo, and vesicles formed connect immediately to physiological membrane-trafficking mechanisms. We suggest that lateral and transbilayer inhomogeneities of the plasma membrane provide potential energies that, when unbridled by triggers, can drive endocytosis by lipidic forces.

scholarly journals Legionella pneumophila LegC7 effector protein drives aberrant ER:endosome fusion in yeast

2020 ◽  
Author(s):  
Nathan K. Glueck ◽  
Kevin M. O’Brien ◽  
Vincent J. Starai
Keyword(s):  
Membrane Trafficking ◽  
Legionella Pneumophila ◽  
Molecular Mechanisms ◽  
Bacterial Pathogen ◽  
Severe Form ◽  
Effector Proteins ◽  
Effector Protein ◽  
Host Membrane ◽  
Membrane Tethering

AbstractLegionella pneumophila is a facultative intracellular bacterial pathogen, causing the severe form of pneumonia known as Legionnaires’ disease. Legionella actively alters host organelle trafficking through the activities of ‘effector’ proteins secreted via a TypeIVB secretion system, in order to construct the bacteria-laden Legionella-containing vacuole (LCV) and prevent lysosomal degradation. The LCV is derived from membrane derived from host ER, secretory vesicles, and phagosomes, although the precise molecular mechanisms that drive its synthesis remain poorly understood. In an effort to characterize the in vivo activity of the LegC7/YlfA SNARE-like effector protein from Legionella in the context of eukaryotic membrane trafficking in yeast, we find that LegC7 interacts with the Emp46p/Emp47p ER-to-Golgi glycoprotein cargo adapter complex, alters ER morphology, and induces aberrant ER:endosome fusion, as measured by visualization of ER cargo degradation, reconstitution of split-GFP proteins, and enhanced oxidation of the ER lumen. LegC7-dependent toxicity, disruption of ER morphology, and ER:endosome fusion events were dependent upon endosomal VPS class C tethering complexes and the endosomal t-SNARE, Pep12p. This work establishes a model in which LegC7 functions to recruit host ER material to the bacterial phagosome during infection by inducing membrane fusion, potentially through interaction with host membrane tethering complexes and/or cargo adapters.

scholarly journals Cdc42 negatively regulates endocytosis during apical plasma membrane maintenance and development in mouse tubular organs in vivo

2018 ◽  
Author(s):  
Akiko Shitara ◽  
Lenka Malec ◽  
Seham Ebrahim ◽  
Desu Chen ◽  
Christopher Bleck ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Fluid Secretion ◽  
Small Gtpase ◽  
Model Systems ◽  
Apical Plasma Membrane ◽  
Adult Mice ◽  
Endocytic Pathways

AbstractLumen establishment and maintenance are fundamental for tubular organs physiological functions. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in mammalian model systems in vivo. Here we used the salivary glands of live mice to examine the role of the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur. Depletion of Cdc42 in adult mice induced a significant expansion of the apical canaliculi, whereas depletion at late embryonic stages resulted in a complete inhibition of their post-natal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and towards the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways.

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