scholarly journals Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry

2009 ◽  
Vol 425 (1) ◽  
pp. 159-168 ◽  
Author(s):  
Ciara M. Walsh ◽  
Michael Chvanov ◽  
Lee P. Haynes ◽  
Ole H. Petersen ◽  
Alexei V. Tepikin ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Phosphatidylinositol 3 Kinase ◽  
Enhanced Yellow Fluorescent Protein ◽  
Store Depletion ◽  
Store Operated Calcium Entry ◽  
Translocation Mechanism ◽  
Direct Binding ◽  
Phosphatidylinositol 4

Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER–PM (endoplasmic reticulum–plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER–PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1–EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 μM) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER–PM junctions with a second translocation mechanism involving direct STIM1–Orai interactions.

scholarly journals GLP-1 Receptor Stimulation of the Lateral Parabrachial Nucleus Reduces Food Intake: Neuroanatomical, Electrophysiological, and Behavioral Evidence

Endocrinology ◽  
2014 ◽  
Vol 155 (11) ◽  
pp. 4356-4367 ◽  
Author(s):  
Jennifer E. Richard ◽  
Imre Farkas ◽  
Fredrik Anesten ◽  
Rozita H. Anderberg ◽  
Suzanne L. Dickson ◽  
...  
Keyword(s):  
Feeding Behavior ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Parabrachial Nucleus ◽  
Lateral Parabrachial Nucleus ◽  
Electrophysiological Studies ◽  
Glucagon Like Peptide 1 ◽  
Behavioral Evidence ◽  
Stimulation Of

Abstract The parabrachial nucleus (PBN) is a key nucleus for the regulation of feeding behavior. Inhibitory inputs from the hypothalamus to the PBN play a crucial role in the normal maintenance of feeding behavior, because their loss leads to starvation. Viscerosensory stimuli result in neuronal activation of the PBN. However, the origin and neurochemical identity of the excitatory neuronal input to the PBN remain largely unexplored. Here, we hypothesize that hindbrain glucagon-like peptide 1 (GLP-1) neurons provide excitatory inputs to the PBN, activation of which may lead to a reduction in feeding behavior. Our data, obtained from mice expressing the yellow fluorescent protein in GLP-1-producing neurons, revealed that hindbrain GLP-1-producing neurons project to the lateral PBN (lPBN). Stimulation of lPBN GLP-1 receptors (GLP-1Rs) reduced the intake of chow and palatable food and decreased body weight in rats. It also activated lPBN neurons, reflected by an increase in the number of c-Fos-positive cells in this region. Further support for an excitatory role of GLP-1 in the PBN is provided by electrophysiological studies showing a remarkable increase in firing of lPBN neurons after Exendin-4 application. We show that within the PBN, GLP-1R activation increased gene expression of 2 energy balance regulating peptides, calcitonin gene-related peptide (CGRP) and IL-6. Moreover, nearly 70% of the lPBN GLP-1 fibers innervated lPBN CGRP neurons. Direct intra-lPBN CGRP application resulted in anorexia. Collectively, our molecular, anatomical, electrophysiological, pharmacological, and behavioral data provide evidence for a functional role of the GLP-1R for feeding control in the PBN.

scholarly journals PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

2021 ◽  
Author(s):  
Max Gass ◽  
Sarah Borkowsky ◽  
Marie-Luise Lotz ◽  
Rita Schroeter ◽  
Pavel Nedvetsky ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Model System ◽  
Dominant Negative ◽  
Slit Diaphragm ◽  
Phosphatidylinositol 3 Kinase ◽  
Ectopic Activation ◽  
Phosphatidylinositol 4 ◽  
Asymmetrically Distributed ◽  
Phosphatidylinositol 3

Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

scholarly journals Genetic and transgenic reagents for Drosophila simulans, D. mauritiana, D. yakuba, D. santomea and D. virilis

2016 ◽  
Author(s):  
David L. Stern ◽  
Justin Crocker ◽  
Yun Ding ◽  
Nicolas Frankel ◽  
Gretchen Kappes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Integration Site ◽  
Yellow Fluorescent Protein ◽  
Model Systems ◽  
Enhanced Yellow Fluorescent Protein ◽  
Functional Studies ◽  
Site Specific Integration ◽  
Genetic Strains ◽  
Fluorescent Molecules ◽  
Integration Efficiency

AbstractSpecies of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. Studies in these species have been limited, however, by a paucity of genetic and transgenic reagents. Here we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein gene expressed in the eyes and a phiC31 attP site-specific integration site. We have tested a subset of these lines for integration efficiency and reporter gene expression levels. We have also generated a smaller collection of other lines expressing other genetically encoded fluorescent molecules in the eyes and a number of other transgenic reagents that will be useful for functional studies in these species. In addition, we have mapped the insertion locations of 58 transposable elements in D. virilis that will be useful for genetic mapping studies.

scholarly journals Pancreatic islet function in a transgenic mouse expressing fluorescent protein

2005 ◽  
Vol 186 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Daniel Nyqvist ◽  
Göran Mattsson ◽  
Martin Köhler ◽  
Varda Lev-Ram ◽  
Arne Andersson ◽  
...  
Keyword(s):  
Pancreatic Islet ◽  
Glucose Homeostasis ◽  
Fluorescent Protein ◽  
Regional Blood Flow ◽  
Animal Health ◽  
Yellow Fluorescent Protein ◽  
Islet Function ◽  
Enhanced Yellow Fluorescent Protein ◽  
Isolated Pancreatic Islets ◽  
Arterial Blood

Pancreatic islet function and glucose homeostasis have been characterized in the transgenic YC-3.0 mouse, which expresses the yellow chameleon 3.0 (YC-3.0) protein under the control of the β-actin and the cytomegalovirus promoters. Fluorescence from the enhanced yellow fluorescent protein (EYFP), one part of the yellow chameleon protein, was used as a reporter of transgene expression. EYFP was expressed in different quantities throughout most cell types, including islet endocrine and stromal cells. No adverse effects of the transgene on animal health, growth or fertility were observed. Likewise, in vivo glucose homeostasis, mean arterial blood pressure and regional blood flow values were normal. Furthermore, the transgenic YC-3.0 mouse had a normal β-cell volume and mass as well as glucose-stimulated insulin release in vitro, compared with the C57BL/6 control mouse. Isolated islets from YC-3.0 animals continuously expressed the transgene and reversed hyperglycemia when transplanted under the renal capsule of alloxan-diabetic nude mice. We conclude that isolated pancreatic islets from YC-3.0 animals implanted into recipients without any EYFP expression, constitute a novel and versatile model for studies of islet engraftment.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Celina Frank ◽  
Dieter Jendrossek
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Eukaryotic Cells ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Polyphosphate Granules

ABSTRACT Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have also been described for Agrobacterium tumefaciens (M. Seufferheld, M. Vieira, A. Ruiz, C. O. Rodrigues, S. Moreno, and R. Docampo, J Biol Chem 278:29971–29978, 2003, https://doi.org/10.1074/jbc.M304548200). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this alphaproteobacterium. Unexpectedly, fluorescence microscopic investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H+-translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located close together, near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1 Δppk2) were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear. IMPORTANCE The uptake of alphaproteobacterium-like cells by ancestors of eukaryotic cells and subsequent conversion of these alphaproteobacterium-like cells to mitochondria are thought to be key steps in the evolution of the first eukaryotic cells. The identification of acidocalcisomes in two alphaproteobacterial species some years ago and the presence of homologs of the vacuolar proton-translocating pyrophosphatase HppA, a marker protein of the acidocalcisome membrane in eukaryotes, in virtually all species within the alphaproteobacteria suggest that eukaryotic acidocalcisomes might also originate from related structures in ancestors of alphaproteobacterial species. Accordingly, alphaproteobacterial acidocalcisomes and eukaryotic acidocalcisomes should have similar features. Since hardly any information is available on bacterial acidocalcisomes, this study aimed at the characterization of organelle-like structures in alphaproteobacterial cells, with A. tumefaciens as an example.

scholarly journals S1P activates store-operated calcium entry via receptor- and non-receptor-mediated pathways in vascular smooth muscle cells

2011 ◽  
Vol 300 (4) ◽  
pp. C919-C926 ◽  
Author(s):  
Kristen Park Hopson ◽  
Jessica Truelove ◽  
Jerold Chun ◽  
Yumei Wang ◽  
Christian Waeber
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Muscle Cells ◽  
Calcium Entry ◽  
Fluorescence Signal ◽  
Store Operated Calcium Entry

Sphingosine-1-phosphate (S1P) has been shown to modulate intracellular Ca2+ through both G protein-coupled receptors and intracellular second messenger pathways. The precise mechanism by which S1P activates store-operated calcium entry (SOCE) in vascular smooth muscle cells (VSMCs) has not been fully characterized. Because sphingolipids and Ca2+ modulate proliferation and constriction in VSMCs, characterizing the connection between S1P and SOCE may provide novel therapeutic targets for vascular diseases. We found that S1P triggered STIM1 puncta formation and SOCE in VSMCs. S1P-activated SOCE was inhibited by 2-aminoethoxydiphenyl borate (2-APB), diethylstilbestrol (DES), and gadolinium (Gd3+). SOCE was observed in VSMCs lacking either S1P2 or S1P3 receptors, suggesting that S1P acts via multiple signaling pathways. Indeed, both extracellular and intracellular S1P application increased the total internal reflection fluorescence signal in VSMCs cells transfected with STIM1-yellow fluorescent protein in a 2-APB-sensitive manner. These data, and the fact that 2-APB, DES, and Gd3+ all inhibited S1P-induced cerebral artery constriction, suggest that SOCE modulates S1P-induced vasoconstriction in vivo. Finally, S1P-induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P1, and S1P3 receptor mRNA. These data demonstrate that S1P can act through both receptors and a novel intracellular pathway to activate SOCE. Because S1P-induced SOCE contributes to vessel constriction and is increased in proliferative VSMCs, it is likely that S1P/SOCE signaling in proliferative VSMCs may play a role in vascular dysfunction such as atherosclerosis and diabetes.

scholarly journals Monitoring Biofilm Formation and Microbial Interactions that May Occur During a Salmonella Contamination Incident across the Network of a Water Bottling Plant

2019 ◽  
Vol 7 (8) ◽  
pp. 236
Author(s):  
Karampoula ◽  
Doulgeraki ◽  
Fotiadis ◽  
Tampakaki ◽  
Nychas
Keyword(s):  
Bacterial Communities ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Yellow Fluorescent Protein ◽  
Foodborne Pathogen ◽  
Microbial Interactions ◽  
Enhanced Yellow Fluorescent Protein ◽  
Serovar Typhimurium ◽  
Gradient Gel Electrophoresis

The present study aims to monitor the ability of Salmonella to colonize and compete as a member of the mixed species biofilm within key points at a water bottling plant, in case of a contamination incident with this major foodborne pathogen. To achieve this goal, bacterial communities throughout the production line were collected and their identities were investigated by microbial counts and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). These bacterial communities alone or along with constructed Salmonella enterica serovar Typhimurium (ST) fluorescence-based bioreporters were left to form a biofilm on stainless steel for 6 days at 20 °C. ST bioreporters were constructed by introducing plasmids expressing EYFP (enhanced yellow fluorescent protein) fusions of the genes csgB, csrA, sspH2, and fliD into ST 14028S. The bead vortexing-plate counting method was applied for the enumeration of the biofilm population, while the behavior of the bioreporters was evaluated by fluorescence microscopy. From a set of 16 samples that were collected from the plant, species of Citrobacter, Staphylococcus, Pseudomonas, Bacillus, and Exiguobacterium were identified. The presence of these indigenous bacteria neither inhibited nor enhanced the biofilm formation of ST in mixed bacterial communities (p > 0.05). Furthermore, the csrA-based bioreporter was shown to be induced in multispecies biofilms with Citrobacter. In conclusion, this study enhanced our knowledge of bacterial interactions occurring within a biofilm in a water bottling plant.

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