The organic anion transport inhibitor, probenecid, inhibits the transport of Lucifer Yellow at the plasma membrane and the tonoplast in suspensioncultured plant cells

1991 ◽  
Vol 99 (3) ◽  
pp. 545-555 ◽  
Author(s):  
LOUISE COLE ◽  
JULIAN COLEMAN ◽  
ANNE KEARNS ◽  
GARETH MORGAN ◽  
CHRIS HAWES
Keyword(s):  
Plasma Membrane ◽  
Lucifer Yellow ◽  
Organic Anion ◽  
Plant Cells ◽  
Fluid Phase ◽  
Cell Suspension Cultures ◽  
Carrot Cells ◽  
Anion Transporters ◽  
Plant Cell Suspension Cultures ◽  
Plant Membranes

In this paper we report on the uptake of the membrane-impermeant fluorescent probe Lucifer Yellow CH (LY-CH) into the vacuolar system of plant cell suspension cultures. LY-CH is internalised into vacuoles of maize cells at a faster ‘rate’ than carrot cells and in each case, the probe is also trapped at the cell wall. In the presence of the uricosuric drug probenecid, the vacuolar uptake of LY-CH by carrot and maize cells is inhibited and in some cells internalisation of probe is blocked at the plasma membrane. In electroporated carrot cells, LY-CH is sequestered slowly from the cytoplasm into vacuoles by a probenecid-inhibitable transport process. These results are compared with the effects of probenecid on the sequestration of LY-CH from the cytoplasm into the lysosomal system of fibroblasts. In view of the above findings and recent evidence for the putative uptake of LY-CH by fluid-phase endocytosis in plant cells, the possibility that LY-CH is transported across plant membranes via probenecidinhibitable organic anion transporters is discussed

scholarly journals TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae

2017 ◽  
Vol 37 (7) ◽  
Author(s):  
Françoise M. Roelants ◽  
Kristin L. Leskoske ◽  
Ross T. A. Pedersen ◽  
Alexander Muir ◽  
Jeffrey M.-H. Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Effector Protein ◽  
Multiple Substrates ◽  
Downstream Effector ◽  
Fluid Phase Endocytosis ◽  
Actin Patch ◽  
Resistance To Doxorubicin

ABSTRACT Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis.

scholarly journals A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages.

1988 ◽  
Vol 107 (3) ◽  
pp. 887-896 ◽  
Author(s):  
T H Steinberg ◽  
J A Swanson ◽  
S C Silverstein
Keyword(s):  
Plasma Membrane ◽  
Lucifer Yellow ◽  
Organic Anion ◽  
Buoyant Density ◽  
Endocytic Pathway ◽  
Cytoplasmic Matrix ◽  
Cytoplasmic Vacuoles ◽  
J774 Cells ◽  
Membrane Bound ◽  
Texas Red

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.

scholarly journals Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits.

1991 ◽  
Vol 114 (3) ◽  
pp. 423-431 ◽  
Author(s):  
C Fuhrer ◽  
I Geffen ◽  
M Spiess
Keyword(s):  
Plasma Membrane ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Membrane Receptors ◽  
Immunogold Localization ◽  
Wild Type ◽  
Coated Pits ◽  
Residual Rate ◽  
Tyrosine Residues ◽  
Plasma Membrane Receptors

The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization was reduced by a factor of four. The residual rate of endocytosis, however, was still significantly higher than that of resident plasma membrane proteins. Upon acidification of the cytoplasm, which specifically inhibits the formation of clathrin-coated vesicles but not uptake of the fluid phase marker Lucifer yellow, residual endocytosis was blocked. By immunoelectron microscopy mutant H1 could be directly demonstrated in coated pits. The fraction of wild-type and mutant H1 present in coated pits as determined by immunogold localization correlated well with the respective rates of internalization. Thus, mutation of tyrosine-5 only partially inactivates recognition of H1 for incorporation into coated pits.

scholarly journals Phospholipid Signaling Is a Component of the Salicylic Acid Response in Plant Cell Suspension Cultures

2020 ◽  
Vol 21 (15) ◽  
pp. 5285
Author(s):  
Beatriz A. Rodas-Junco ◽  
Geovanny I. Nic-Can ◽  
Armando Muñoz-Sánchez ◽  
S. M. Teresa Hernández-Sotomayor
Keyword(s):  
Salicylic Acid ◽  
Phospholipase D ◽  
Cellular Response ◽  
Plant Cells ◽  
Cell Suspension Cultures ◽  
Plant Cell Suspension Cultures ◽  
Plant Cell Suspension ◽  
Phospholipid Signaling ◽  
Sa Signaling

Salicylic acid (SA) is an important signaling molecule involved in plant defense. While many proteins play essential roles in SA signaling, increasing evidence shows that responses to SA appear to involve and require lipid signals. The phospholipid-generated signal transduction involves a family of enzymes that catalyze the hydrolysis or phosphorylation of phospholipids in membranes to generate signaling molecules, which are important in the plant cellular response. In this review, we focus first, the role of SA as a mitigator in biotic/abiotic stress. Later, we describe the experimental evidence supporting the phospholipid–SA connection in plant cells, emphasizing the roles of the secondary lipid messengers (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA)) and related enzymes (phospholipase D (PLD) and phospholipase C (PLC)). By placing these recent finding in context of phospholipids and SA in plant cells, we highlight the role of phospholipids as modulators in the early steps of SA triggered transduction in plant cells.

scholarly journals Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix.

1987 ◽  
Vol 105 (6) ◽  
pp. 2695-2702 ◽  
Author(s):  
T H Steinberg ◽  
A S Newman ◽  
J A Swanson ◽  
S C Silverstein
Keyword(s):  
Fluorescent Dyes ◽  
Lucifer Yellow ◽  
Organic Anion ◽  
Peritoneal Macrophages ◽  
Organic Anions ◽  
Organic Anion Transporters ◽  
Extracellular Medium ◽  
Cytoplasmic Matrix ◽  
Anion Transporters ◽  
Cytoplasmic Vacuoles

We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy-requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p-[dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages.

scholarly journals Endotransglycosylation of xyloglucans in plant cell suspension cultures

1991 ◽  
Vol 279 (2) ◽  
pp. 529-535 ◽  
Author(s):  
R C Smith ◽  
S C Fry
Keyword(s):  
Plant Cell ◽  
Trichloroacetic Acid ◽  
Plant Cells ◽  
Cell Suspension Cultures ◽  
Primary Cell Wall ◽  
Extracellular Polymers ◽  
Plant Cell Suspension Cultures ◽  
Plant Cell Suspension ◽  
Wall Loosening

1. A xyloglucan-derived nonasaccharide ([3H]XG9; Glc4,Xyl3,Gal,Fuc) was neither taken up by cultured plant cells nor appreciably hydrolysed by them, but a proportion of it became incorporated into extracellular polymers in all cultures tested (Spinacia, Daucus, Rosa, Acer, Capsicum, Zea and Festuca). 2. In Spinacia these polymers were soluble in 20% (w/v) trichloroacetic acid, had apparent Mr 20,000-30,000, were able to bind reversibly to cellulose powder and were susceptible to hydrolysis by endo-beta-(1→4)-D-glucanase, indicating that they were xyloglucans. 3. The linkage formed between [3H]XG9 and the xyloglucan was alkali-stable and glucanase-labile, indicating that the reaction responsible for the incorporation was a transglycosylation. 4. The reducing terminus of the XG9 moiety remained reducing (convertible into [3H]glucitol by NaBH4) after incorporation into the polymer, showing that the XG9 was the glycosyl acceptor and the polysaccharide the donor. 5. The results provide the first evidence that polymeric xyloglucans are subject in vivo to cleavage followed by transfer of the cut end on the other xyloglucan-related molecules. 6. Similar endotransglycosylation reactions could occur within the primary cell wall, between pairs of high-Mr structural xyloglucan molecules. Such a reaction would provide a mechanism for reversible wall loosening and may thus be relevant to our understanding of plant cell growth.

Enrichment of vitronectin- and fibronectin-like proteins in NaCI-adapted plant cells and evidence for their involvement in plasma membrane-cell wall adhesion

1993 ◽  
Vol 3 (5) ◽  
pp. 637-646 ◽  
Author(s):  
Jian-Kang Zhu ◽  
Jun Shi ◽  
Utpal Singh ◽  
Sarah E. Wyatt ◽  
Ray A. Bressan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Plant Cells ◽  
Membrane Cell

Multimodal Atomic Force Imaging of Open Hemichannelinduced Modulation of Cell Volume and Viscoelastic Properties

1999 ◽  
Vol 5 (S2) ◽  
pp. 998-999
Author(s):  
Seung K. Rhee ◽  
Arjan P. Quist ◽  
Hai Lin ◽  
Nils Almqvist ◽  
Ratneshx Lai
Keyword(s):  
Plasma Membrane ◽  
Cell Volume ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Dye Uptake ◽  
Aortic Endothelial Cells ◽  
Atomic Force ◽  
Cell Plasma ◽  
Uptake Assay ◽  
Gap Junctional

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.

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