scholarly journals Fast Diffusion Sustains Plasma Membrane Accumulation of Phosphatase of Regenerating Liver-1

2020 ◽  
Vol 8 ◽  
Author(s):  
Patricia Castro-Sánchez ◽  
Sara Hernández-Pérez ◽  
Oscar Aguilar-Sopeña ◽  
Rocia Ramírez-Muñoz ◽  
Sandra Rodríguez-Perales ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Electrostatic Interactions ◽  
Fluorescent Protein ◽  
Membrane Lipids ◽  
Correlation Spectroscopy ◽  
Fast Diffusion ◽  
Regenerating Liver ◽  
Recycling Endosomes ◽  
Endogenous Expression ◽  
Phosphatase Of Regenerating Liver

It has been proposed that the accumulation of farnesylated phosphatase of regenerating liver-1 (PRL-1) at the plasma membrane is mediated by static electrostatic interactions of a polybasic region with acidic membrane lipids and assisted by oligomerization. Nonetheless, localization at early and recycling endosomes suggests that the recycling compartment might also contribute to its plasma membrane accumulation. Here, we investigated in live cells the dynamics of PRL-1 fused to the green fluorescent protein (GFP-PRL-1). Blocking the secretory pathway and photobleaching techniques suggested that plasma membrane accumulation of PRL-1 was not sustained by recycling endosomes but by a dynamic exchange of diffusible protein pools. Consistent with this idea, fluorescence correlation spectroscopy in cells overexpressing wild type or monomeric mutants of GFP-PRL-1 measured cytosolic and membrane-diffusing pools of protein that were not dependent on oligomerization. Endogenous expression of GFP-PRL-1 by CRISPR/Cas9 genome edition confirmed the existence of fast diffusing cytosolic and membrane pools of protein. We propose that plasma membrane PRL-1 replenishment is independent of the recycling compartment and the oligomerization state and mainly driven by fast diffusion of the cytosolic pool.

scholarly journals Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

2011 ◽  
Vol 22 (18) ◽  
pp. 3498-3507 ◽  
Author(s):  
Urszula Golebiewska ◽  
Jason G. Kay ◽  
Thomas Masters ◽  
Sergio Grinstein ◽  
Wonpil Im ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Surface Concentration ◽  
Correlation Spectroscopy ◽  
Similar Data ◽  
Fluorescence Correlation ◽  
The Many ◽  
Green Fluorescent

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

scholarly journals Dynamics of a Chemoattractant Receptor in Living Neutrophils during Chemotaxis

1999 ◽  
Vol 10 (4) ◽  
pp. 1163-1178 ◽  
Author(s):  
Guy Servant ◽  
Orion D. Weiner ◽  
Enid R. Neptune ◽  
John W. Sedat ◽  
Henry R. Bourne
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Membrane Lipids ◽  
Leukemia Cell ◽  
Leading Edge ◽  
Relative Strength ◽  
Leukemia Cell Line ◽  
Anterior Pole ◽  
Apparent Increase ◽  
Green Fluorescent

Persistent directional movement of neutrophils in shallow chemotactic gradients raises the possibility that cells can increase their sensitivity to the chemotactic signal at the front, relative to the back. Redistribution of chemoattractant receptors to the anterior pole of a polarized neutrophil could impose asymmetric sensitivity by increasing the relative strength of detected signals at the cell’s leading edge. Previous experiments have produced contradictory observations with respect to receptor location in moving neutrophils. To visualize a chemoattractant receptor directly during chemotaxis, we expressed a green fluorescent protein (GFP)-tagged receptor for a complement component, C5a, in a leukemia cell line, PLB-985. Differentiated PLB-985 cells, like neutrophils, adhere, spread, and polarize in response to a uniform concentration of chemoattractant, and orient and crawl toward a micropipette containing chemoattractant. Recorded in living cells, fluorescence of the tagged receptor, C5aR–GFP, shows no apparent increase anywhere on the plasma membrane of polarized and moving cells, even at the leading edge. During chemotaxis, however, some cells do exhibit increased amounts of highly folded plasma membrane at the leading edge, as detected by a fluorescent probe for membrane lipids; this is accompanied by an apparent increase of C5aR–GFP fluorescence, which is directly proportional to the accumulation of plasma membrane. Thus neutrophils do not actively concentrate chemoattractant receptors at the leading edge during chemotaxis, although asymmetrical distribution of membrane may enrich receptor number, relative to adjacent cytoplasmic volume, at the anterior pole of some polarized cells. This enrichment could help to maintain persistent migration in a shallow gradient of chemoattractant.

Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells

2021 ◽  
Vol 118 (49) ◽  
pp. e2025748118
Author(s):  
Lei Wan ◽  
Xi Chen ◽  
Wolfhard Almers
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Membrane Lipids ◽  
Specific Interaction ◽  
Salt Bridges ◽  
Syntaxin 1A ◽  
Dense Cluster ◽  
Low Levels ◽  
Green Fluorescent

Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules (“on-granule clusters”). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin’s Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

Human Na+/H+ exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria

2002 ◽  
Vol 282 (5) ◽  
pp. C1031-C1041 ◽  
Author(s):  
Christopher L. Brett ◽  
Ying Wei ◽  
Mark Donowitz ◽  
Rajini Rao
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Phylogenetic Analyses ◽  
Human Homolog ◽  
Recycling Endosomes ◽  
Mitochondrial Markers ◽  
Vacuolar Compartment ◽  
Green Fluorescent ◽  
Golgi Network

Since the discovery of the first intracellular Na+/H+exchanger in yeast, Nhx1, multiple homologs have been cloned and characterized in plants. Together, studies in these organisms demonstrate that Nhx1 is located in the prevacuolar/vacuolar compartment of cells where it sequesters Na+ into the vacuole, regulates intravesicular pH, and contributes to vacuolar biogenesis. In contrast, the human homolog of Nhx1, Na+/H+ exchanger isoform 6 (NHE6), has been reported to localize to mitochondria when transiently expressed as a fusion with green fluorescent protein. This result warrants reevaluation because it conflicts with predictions from phylogenetic analyses. Here we demonstrate that when epitope-tagged NHE6 is transiently expressed in cultured mammalian cells, it does not colocalize with mitochondrial markers. It also does not colocalize with markers of the lysosome, late endosome, trans-Golgi network, or Golgi cisternae. Rather, NHE6 is distributed in recycling compartments and transiently appears on the plasma membrane. These results suggest that, like its homologs in yeast and plants, NHE6 is an endosomal Na+/H+ exchanger that may regulate intravesicular pH and volume and contribute to lysosomal biogenesis.

scholarly journals Molecular Clustering of STIM1 with Orai1/CRACM1 at the Plasma Membrane Depends Dynamically on Depletion of Ca2+ Stores and on Electrostatic Interactions

2009 ◽  
Vol 20 (1) ◽  
pp. 389-399 ◽  
Author(s):  
Nathaniel Calloway ◽  
Monika Vig ◽  
Jean-Pierre Kinet ◽  
David Holowka ◽  
Barbara Baird
Keyword(s):  
Plasma Membrane ◽  
Electrostatic Interactions ◽  
Fluorescent Protein ◽  
Immunoglobulin E ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Store Depletion ◽  
Green Fluorescent ◽  
Molecular Clustering ◽  
Micrometer Scale

Activation of store operated Ca2+ entry involves stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER), and calcium channel subunit (Orai1/CRACM1), localized to the plasma membrane. Confocal microscopy shows that thapsigargin-mediated depletion of ER Ca2+ stores in RBL mast cells causes a redistribution of STIM1, labeled with monomeric red fluorescent protein (mRFP), to micrometer-scale ER-plasma membrane junctions that contain Orai1/CRACM1, labeled with monomeric Aequorea coerulescens green fluorescent protein (AcGFP). Using fluorescence resonance energy transfer (FRET), we determine that this visualized coredistribution is accompanied by nanoscale interaction of STIM1-mRFP and AcGFP-Orai1/CRACM1. We find that antigen stimulation of immunoglobulin E receptors causes much less Orai1/CRACM1 and STIM1 association, but strong interaction is observed under conditions that prevent refilling of ER stores. Stimulated association monitored by FRET is inhibited by sphingosine derivatives in parallel with inhibition of Ca2+ influx. Similar structural and functional effects are caused by mutation of acidic residues in the cytoplasmic segment of Orai1/CRACM1, suggesting a role for electrostatic interactions via these residues in the coupling of Orai1/CRACM1 to STIM1. Our results reveal dynamic molecular interactions between STIM1 and Orai1/CRACM1 that depend quantitatively on electrostatic interactions and on the extent of store depletion.

Elevated cAMP increases aquaporin-3 plasma membrane diffusion

2014 ◽  
Vol 306 (6) ◽  
pp. C598-C606 ◽  
Author(s):  
Saw Marlar ◽  
Eva C. Arnspang ◽  
Jennifer S. Koffman ◽  
Else-Merete Løcke ◽  
Birgitte M. Christensen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Collecting Duct ◽  
Urine Concentration ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Egfp Expression ◽  
Membrane Diffusion ◽  
Short Term ◽  
Expression Levels

Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water exits via basolateral AQP3 and AQP4. Upon long-term stimulation with AVP or during thirst, expression levels of both AQP2 and AQP3 are increased; however, there is so far no evidence for short-term AVP regulation of AQP3 or AQP4. To facilitate the increase in transepithelial water transport, AQP3 may be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts, and/or changes in steady-state turnover, which could result in changes in the diffusion behavior of AQP3. Thus we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin to reveal if AQP3 could be short-term regulated by AVP. k-Space image correlation spectroscopy (kICS) analysis of time-lapse image sequences of basolateral enhanced green fluorescent protein-tagged AQP3 (AQP3-EGFP) revealed that the forskolin-mediated elevation of cAMP increased the diffusion coefficient by 58% from 0.0147 ± 0.0082 μm2/s (control) to 0.0232 ± 0.0085 μm2/s (forskolin, P < 0.05). Quantum dot-conjugated antibody labeling also revealed a significant increase in AQP3 diffusion upon forskolin treatment by 44% [0.0104 ± 0.0040 μm2/s (control) vs. 0.0150 ± 0.0016 μm2/s (forskolin, P < 0.05)]. Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption.

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.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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