scholarly journals Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity

2001 ◽  
Vol 154 (2) ◽  
pp. 355-368 ◽  
Author(s):  
Kristina D. Micheva ◽  
Ronald W. Holz ◽  
Stephen J. Smith
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Confocal Imaging ◽  
Resting Cells ◽  
Ph Domain ◽  
Cellular Processes ◽  
Vesicle Recycling ◽  
Green Fluorescent

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.

scholarly journals Oscillations of Phospholipase C Activity Triggered by Depolarization and Ca2+Influx in Insulin-secreting Cells

2004 ◽  
Vol 279 (19) ◽  
pp. 19396-19400 ◽  
Author(s):  
Sophia Thore ◽  
Oleg Dyachok ◽  
Anders Tengholm
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Evanescent Wave ◽  
Fluorescent Protein ◽  
Purinergic Receptor ◽  
Dynamic Control ◽  
Pleckstrin Homology Domain ◽  
Resting Cells ◽  
Cellular Processes ◽  
Green Fluorescent

Phospholipase C (PLC) is a ubiquitous enzyme involved in the regulation of a variety of cellular processes. Its dependence on Ca2+is well recognized, but it is not known how PLC activity is affected by physiological variations of the cytoplasmic Ca2+concentration ([Ca2+]i). Here, we applied evanescent wave microscopy to monitor PLC activity in parallel with [Ca2+]iin individual insulin-secreting INS-1 cells using the phosphatidylinositol 4,5-bisphosphate- and inositol 1,4,5-trisphosphate-binding pleckstrin homology domain from PLCδ1fused to green fluorescent protein (PHPLCδ1-GFP) and the Ca2+indicator fura red. In resting cells, PHPLCδ1-GFP was located predominantly at the plasma membrane. Activation of PLC by muscarinic or purinergic receptor stimulation resulted in PHPLCδ1-GFP translocation from the plasma membrane to the cytoplasm, detected as a decrease in evanescent wave-excited PHPLCδ1-GFP fluorescence. Using this translocation as a measure of PLC activity, we found that depolarization by raising extracellular [K+] triggered activation of the enzyme. This effect could be attributed both to a rise of [Ca2+]iand to depolarizationper se, because some translocation persisted during depolarization in a Ca2+-deficient medium containing the Ca2+chelator EGTA. Moreover, oscillations of [Ca2+]iresulting from depolarization with Ca2+influx evoked concentration-dependent periodic activation of PLC. We conclude that PLC activity is under tight dynamic control of [Ca2+]i. In insulin-secreting β-cells, this mechanism provides a link between Ca2+influx and release from intracellular stores that may be important in the regulation of insulin secretion.

scholarly journals Phosphatidylinositol-4,5-Bisphosphate-Rich Plasma Membrane Patches Organize Active Zones of Endocytosis and Ruffling in Cultured Adipocytes

2004 ◽  
Vol 24 (20) ◽  
pp. 9102-9123 ◽  
Author(s):  
Shaohui Huang ◽  
Larry Lifshitz ◽  
Varsha Patki-Kamath ◽  
Richard Tuft ◽  
Kevin Fogarty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Actin Dynamics ◽  
Ph Domain ◽  
Membrane Ruffling ◽  
Green Fluorescent

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

scholarly journals The Yeast Inositol Polyphosphate 5-Phosphatases Inp52p and Inp53p Translocate to Actin Patches following Hyperosmotic Stress: Mechanism for Regulating Phosphatidylinositol 4,5-Bisphosphate at Plasma Membrane Invaginations

2000 ◽  
Vol 20 (24) ◽  
pp. 9376-9390 ◽  
Author(s):  
Lisa M. Ooms ◽  
Brad K. McColl ◽  
Fenny Wiradjaja ◽  
A. P. W. Wijayaratnam ◽  
Paul Gleeson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Hyperosmotic Stress ◽  
Resting Cells ◽  
Inositol Polyphosphate ◽  
Rhodamine Phalloidin ◽  
Phosphatase Domain ◽  
Rich Domain ◽  
Green Fluorescent

ABSTRACT The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and PtdIns(3,5)P2. Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P2 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P2 and PtdIns(4,5)P2 at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.

scholarly journals Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

1998 ◽  
Vol 140 (3) ◽  
pp. 659-674 ◽  
Author(s):  
Takao Nakata ◽  
Sumio Terada ◽  
Nobutaka Hirokawa
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Fluorescent Protein ◽  
Plasma Membrane Proteins ◽  
Trans Golgi Network ◽  
Direction Of Movement ◽  
Green Fluorescent ◽  
Golgi Network

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

GABAC ρ1 Subunits Form Functional Receptors But Not Functional Synapses in Hippocampal Neurons

2001 ◽  
Vol 86 (5) ◽  
pp. 2605-2615 ◽  
Author(s):  
Qing Cheng ◽  
Paul M. Burkat ◽  
John C. Kulli ◽  
Jay Yang
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Short Pulse ◽  
Gaba Release ◽  
Ruthenium Red ◽  
Confocal Imaging ◽  
Imaging Data ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent

The ability to control the physiological and pharmacological properties of synaptic receptors is a powerful tool for studying neuronal function and may be of therapeutic utility. We designed a recombinant adenovirus to deliver either a GABAC receptor ρ1 subunit or a mutant GABAA receptor β2 subunit lacking picrotoxin sensitivity [β2(mut)] to hippocampal neurons. A green fluorescent protein (GFP) reporter molecule was simultaneously expressed. Whole cell patch-clamp recordings demonstrated somatic expression of both bicuculline-resistant GABAC receptor-mediated and picrotoxin-resistant GABAA receptor-mediated GABA-evoked currents in ρ1- and β2(mut)-transduced hippocampal neurons, respectively. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of 6-cyano-7-nitroquinoxalene-2,3-dione, Mg2+, and TTX revealed synaptic events with monoexponential activation and biexponential decay phases. Despite the robust expression of somatic GABAC receptors in ρ1-neurons, no bicuculline-resistant mIPSCs were observed. This suggested either a kinetic mismatch between the relatively brief presynaptic GABA release and slow-activating ρ1 receptors or failure of the ρ1 subunit to target properly to the subsynaptic membrane. Addition of ruthenium red, a presynaptic release enhancer, failed to unmask GABACreceptor-mediated mIPSCs. Short pulse (2 ms) application of 1 mM GABA to excised outside-out patches from ρ1 neurons proved that a brief GABA transient is sufficient to activate ρ1 receptors. The simulated-IPSC experiment strongly suggests that if postsynaptic GABACreceptors were present, bicuculline-resistant mIPSCs would have been observed. In contrast, in β2(mut)-transduced neurons, picrotoxin-resistant mIPSCs were observed; they exhibited a smaller peak amplitude and faster decay compared with control. Confocal imaging of transduced neurons revealed ρ1immunofluorescence restricted to the soma, whereas punctate β2(mut) immunofluorescence was seen throughout the neuron, including the dendrites. Together, the electrophysiological and imaging data show that despite robust somatic expression of the ρ1 subunit, the GABACreceptor fails to be delivered to the subsynaptic target. On the other hand, the successful incorporation of β2(mut) subunits into subsynaptic GABAA receptors demonstrates that viral transduction is a powerful method for altering the physiological properties of synapses.

scholarly journals A Conserved Mechanism of Synaptogyrin Localization

2001 ◽  
Vol 12 (8) ◽  
pp. 2275-2289 ◽  
Author(s):  
Hongjuan Zhao ◽  
Michael L. Nonet
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
C Elegans ◽  
C Terminus ◽  
Green Fluorescent ◽  
Cultured Hippocampal Neurons

We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-taggedCaenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates.

scholarly journals Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1

1999 ◽  
Vol 337 (3) ◽  
pp. 575-583 ◽  
Author(s):  
Richard A. CURRIE ◽  
Kay S. WALKER ◽  
Alex GRAY ◽  
Maria DEAK ◽  
Antonio CASAMAYOR ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Lipid Vesicles ◽  
Live Cells ◽  
Ph Domain ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Green Fluorescent

3-Phosphoinositide-dependent protein kinase-1 (PDK1) interacts stereoselectively with the d-enantiomer of PtdIns(3,4,5)P3 (KD 1.6 nM) and PtdIns(3,4)P2 (KD 5.2 nM), but binds with lower affinity to PtdIns3P or PtdIns(4,5)P2. The binding of PtdIns(3,4,5)P3 to PDK1 was greatly decreased by making specific mutations in the pleckstrin homology (PH) domain of PDK1 or by deleting it. The same mutations also greatly decreased the rate at which PDK1 activated protein kinase Bα (PKBα) in vitro in the presence of lipid vesicles containing PtdIns(3,4,5)P3, but did not affect the rate at which PDK1 activated a PKBα mutant lacking the PH domain in the absence of PtdIns(3,4,5)P3. When overexpressed in 293 or PAE cells, PDK1 was located at the plasma membrane and in the cytosol, but was excluded from the nucleus. Mutations that disrupted the interaction of PtdIns(3,4,5)P3 or PtdIns(4,5)P2 with PDK1 abolished the association of PDK1 with the plasma membrane. Growth-factor stimulation promoted the translocation of transfected PKBα to the plasma membrane, but had no effect on the subcellular distribution of PDK1 as judged by immunoelectron microscopy of fixed cells. This conclusion was also supported by confocal microscopy of green fluorescent protein–PDK1 in live cells. These results, together with previous observations, indicate that PtdIns(3,4,5)P3 plays several roles in the PDK1-induced activation of PKBα. First, it binds to the PH domain of PKB, altering its conformation so that it can be activated by PDK1. Secondly, interaction with PtdIns(3,4,5)P3 recruits PKB to the plasma membrane with which PDK1 is localized constitutively by virtue of its much stronger interaction with PtdIns(3,4,5)P3 or PtdIns(4,5)P2. Thirdly, the interaction of PDK1 with PtdIns(3,4,5)P3 facilitates the rate at which it can activate PKB.

Intracellular trafficking/membrane targeting of human reduced folate carrier expressed in Xenopus oocytes

2001 ◽  
Vol 281 (6) ◽  
pp. G1477-G1486 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Ian Parker ◽  
Hamid M. Said
Keyword(s):  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Fluorescent Protein ◽  
Cytoplasmic Tail ◽  
Confocal Imaging ◽  
Membrane Targeting ◽  
Reduced Folate Carrier ◽  
Animal Pole ◽  
Molecular Determinant ◽  
Green Fluorescent

The major cellular pathway for uptake of the vitamin folic acid, including its absorption in the intestine, is via a plasma membrane carrier system, the reduced folate carrier (RFC). Very little is known about the mechanisms that control intracellular trafficking and plasma membrane targeting of RFC. To begin addressing these issues, we used Xenopus oocyte as a model system and examined whether the signal that targets the protein to the plasma membrane is located in the COOH-terminal cytoplasmic tail or in the backbone of the polypeptide. We also examined the role of microtubules and microfilaments in intracellular trafficking of the protein. Confocal imaging of human RFC (hRFC) fused to the enhanced green fluorescent protein (hRFC-EGFP) showed that the protein was expressed at the plasma membrane, with expression confined almost entirely to the animal pole of the oocyte. Localization of hRFC at the plasma membrane was not affected by partial or total truncation of the COOH-terminal tail of the polypeptide, whereas a construct of the cytoplasmic tail fused to EGFP was not found at the plasma membrane. Disruption of microtubules, but not microfilaments, prevented hRFC expression at the plasma membrane. These results demonstrate that the molecular determinant(s) that directs plasma membrane targeting of hRFC is located within the backbone of the polypeptide and that intact microtubules, but not microfilaments, are essential for intracellular trafficking of the protein.

scholarly journals PLCζ causes Ca2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P2

2012 ◽  
Vol 23 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Yuansong Yu ◽  
Michail Nomikos ◽  
Maria Theodoridou ◽  
George Nounesis ◽  
F. Anthony Lai ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Inositol Polyphosphate ◽  
Ph Domain ◽  
Phosphoinositide Signaling ◽  
Intracellular Ca ◽  
Mammalian Fertilization ◽  
Green Fluorescent ◽  
Mammalian Eggs ◽  
Mouse Eggs

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca2+ oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate–mediated Ca2+ release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P2 targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P2 with a green fluorescent protein–tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P2 from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca2+ oscillations. Specific depletion of this PI(4,5)P2 pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca2+ oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P2, PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P2 after PLCζ injection. Targeted depletion of vesicular PI(4,5)P2 by expression of ciPLCζ-fused Inp54p inhibited the Ca2+ oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca2+ mobilization by hydrolyzing internal PI(4,5)P2 stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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