Uptake of Lucifer Yellow CH into intact barley roots: Evidence for fluid-phase endocytosis

Planta ◽  
1988 ◽  
Vol 176 (4) ◽  
pp. 541-547 ◽  
Author(s):  
K. J. Oparka ◽  
D. Robinson ◽  
D. A. M. Prior ◽  
P. Derrick ◽  
K. M. Wright
Keyword(s):  
Lucifer Yellow ◽  
Fluid Phase ◽  
Lucifer Yellow Ch ◽  
Fluid Phase Endocytosis

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 Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran.

1987 ◽  
Vol 105 (5) ◽  
pp. 1981-1987 ◽  
Author(s):  
R A Preston ◽  
R F Murphy ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Lucifer Yellow ◽  
Ph Dependence ◽  
Fluid Phase ◽  
Fluorescein Isothiocyanate ◽  
Low Molecular Weight ◽  
Yeast Saccharomyces Cerevisiae ◽  
Fluid Phase Endocytosis ◽  
Rapid Kinetics

Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants. After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.

Is fluid-phase endocytosis conserved in hepatocytes of species acclimated and adapted to different temperatures?

2000 ◽  
Vol 278 (2) ◽  
pp. R529-R536 ◽  
Author(s):  
David Padrón ◽  
Michael E. Bizeau ◽  
Jeffrey R. Hazel
Keyword(s):  
Lucifer Yellow ◽  
Fluid Phase ◽  
Membrane Lipid ◽  
Primary Objective ◽  
Chain Polyunsaturated Fatty Acid ◽  
Chow Diet ◽  
Sprague Dawley ◽  
Fluid Phase Endocytosis ◽  
Different Temperatures ◽  
Long Chain

Our primary objective was to determine if rates of fluid-phase endocytosis (FPE) were conserved in hepatocytes from organisms acclimated and adapted to different temperatures. To this aim, the fluorescent dye Lucifer yellow was employed to measure FPE at different assay temperatures (AT) in hepatocytes from 5°C- and 20°C-acclimated trout, Oncorhynchus mykiss (at 5 and 20°C AT), 22°C- and 35°C-acclimated tilapia, Oreochromis nilotica (at 22 and 35°C AT), and the Sprague-Dawley rat (at 10, 20, and 37°C AT). FPE was also studied in rats fed a long-chain polyunsaturated fatty acid (PUFA)-enriched diet (at 10°C AT). Despite being temperature dependent, endocytic rates (values in pl ⋅ cell− 1 ⋅ h− 1) in both species of fish were compensated after a period of acclimation. For example, in 20°C-acclimated trout, the rate of endocytosis declined from 1.84 to 1.07 when the AT was reduced from 20 to 5°C; however, after a period of acclimation at 5°C, the rate (at 5°C AT) was largely restored (1.80) and almost perfectly compensated (95%). In tilapia, endocytic rates were also temperature compensated, although only partially (36%). Relatively similar rates obtained at 5°C in 5°C-acclimated trout (1.8), at 20°C in 20°C-acclimated trout (1.84), and at 22°C in 22°C-acclimated tilapia (2.2) suggest that endocytic rates are somewhat conserved in these two species of fish. In contrast, the rate in rat measured at 37°C (16.83) was severalfold greater than in fish at their respective body temperatures. A role for lipids in determining rates of endocytosis was supported by data obtained at 10°C in hepatocytes isolated from rats fed a long-chain PUFA-enriched diet: endocytic rates were higher (5.35 pl ⋅ cell− 1 ⋅ h− 1) than those of rats fed a standard chow diet (2.33 pl ⋅ cell− 1 ⋅ h− 1). The conservation of endocytic rates in fish may be related to their ability to conserve other membrane characteristics (i.e., order or phase behavior) by restructuring their membrane lipid composition or by modulating the activities of proteins that regulate endocytosis and membrane traffic, whereas the lack of conservation between fish and rat may be due to differences in metabolic rate.

scholarly journals Angiotensin Peptide Regulation of Fluid-Phase Endocytosis in Brain Microvessel Endothelial Cell Monolayers

1990 ◽  
Vol 10 (6) ◽  
pp. 827-834 ◽  
Author(s):  
François L. Guillot ◽  
Kenneth L. Audus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Lucifer Yellow ◽  
Primary Cultures ◽  
Fluid Phase ◽  
Ang Ii ◽  
Microvessel Endothelial Cell ◽  
Fluid Phase Endocytosis ◽  
Microvessel Endothelial Cells ◽  
Brain Microvessel

An in vitro model comprised of primary cultures of brain microvessel endothelial cells was used to investigate angiotensin II (Ang II) effects on blood–brain barrier fluid-phase endocytosis. The effects of Ang II, saralasin, sarathrin, bradykinin (BK), and phorbol myristate acetate (PMA) on brain microvessel endothelial cell fluid-phase endocytosis were determined using the fluorescent marker, Lucifer yellow. Nanomolar concentrations of saralasin (a partial Ang II agonist) stimulated brain microvessel endothelial cell endocytosis by 30% whereas Ang II treatment enhanced Lucifer yellow uptake by 20%. Sarathrin (an Ang II antagonist) had no effect on Lucifer yellow uptake. Nanomolar concentrations of BK and PMA also stimulated Lucifer yellow uptake by the brain microvessel endothelial cell by 40 and 95%, respectively. Stimulatory effects of Ang II and saralasin on Lucifer yellow uptake by brain microvessel endothelial cells could be completely blocked by pretreatment with either sarathrin or indomethacin (an inhibitor of prostaglandin synthesis). In contrast, the effects of neither BK nor PMA on brain microvessel endothelial cell uptake of Lucifer yellow were altered by indomethacin pretreatment. Results indicated that Ang II, saralasin, BK, and PMA produce similar stimulatory effects on brain microvessel endothelial cell fluid-phase endocytosis with only Ang II and saralasin, producing increases in brain microvessel endothelial cell fluid-phase endocytosis that appeared to be mediated by prostaglandins.

scholarly journals end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (1) ◽  
pp. 55-65 ◽  
Author(s):  
S Raths ◽  
J Rohrer ◽  
F Crausaz ◽  
H Riezman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Cell Surface Receptor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Fluid Phase Endocytosis ◽  
Alpha Factor

alpha-factor, one of two peptide hormones responsible for synchronized mating between MATa and MAT alpha-cell types in Saccharomyces cerevisiae, binds to its cell surface receptor and is internalized in a time-, temperature-, and energy-dependent manner (Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). After internalization, alpha-factor is delivered to the vacuole via vesicular intermediates and degraded there consistent with an endocytic mechanism (Singer, B., and H. Riezman. 1990. J. Cell Biol. 110:1911-1922; Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). We have isolated two mutants that are defective in the internalization process. Both mutations confer a recessive, temperature-sensitive growth phenotype upon cells that cosegregates with their endocytosis defect. Lucifer yellow, a marker for fluid-phase endocytosis, shows accumulation characteristics in the mutants that are similar to the uptake characteristics of 35S-alpha-factor. The endocytic defect in end4 cells appears immediately upon shift to restrictive temperature and is reversible at permissive temperature if new protein synthesis is allowed. Furthermore, the end4 mutation only affects alpha-factor internalization and not the later delivery of alpha-factor to the vacuole. Other vesicle-mediated processes seem to be normal in end3 and end4 mutants. END3 and END4 are the first genes shown to be necessary for the internalization step of receptor-borne and fluid-phase markers in yeast.

scholarly journals Development of a probenecid-sensitive Lucifer Yellow transport system in vacuolating oat aleurone protoplasts

1992 ◽  
Vol 102 (1) ◽  
pp. 133-139 ◽  
Author(s):  
K. M. WRIGHT ◽  
T. G. E. DAVIES ◽  
S. H. STEELE ◽  
R. A. LEIGH ◽  
K. J. OPARKA
Keyword(s):  
Developmental Stages ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Transport Systems ◽  
Uptake System ◽  
Cytoplasmic Staining ◽  
Simple Diffusion ◽  
Fluid Phase Endocytosis ◽  
Membrane Transport Systems ◽  
Early Developmental

Oat aleurone protoplasts, maintained in liquid culture over a period of five days, have been shown to develop an uptake system capable of transporting the membrane-impermeant probe Lucifer Yellow CH (LYCH). The dye was completely excluded from the densely cytoplasmic, early developmental stages but its uptake increased exponentially after the protoplasts had been cultured for between 2 and 5 days. Culturing induced vacuolation and uptake of the dye was largely restricted to highly vacuolate protoplasts. No cytoplasmic staining was evident. In vacuolate protoplasts uptake was linear with time and saturated with increasing substrate concentration. Low temperature, and addition of the drug probenecid to the incubation medium, completely eliminated LYCH uptake. In contrast to unconjugated LYCH, LYCH-dextrans (Mr 10,000 and 40,000) were excluded from the protoplasts. The data negate simple diffusion and fluid-phase endocytosis as possible candidates for dye uptake and suggest, instead, the development of highly co-ordinated membrane transport systems on both plasmalemma and tonoplast.

scholarly journals Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages.

1985 ◽  
Vol 100 (3) ◽  
pp. 851-859 ◽  
Author(s):  
J A Swanson ◽  
B D Yirinec ◽  
S C Silverstein
Keyword(s):  
Steady State ◽  
Horseradish Peroxidase ◽  
Phorbol Esters ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Peritoneal Macrophages ◽  
Tumor Promoter ◽  
Steady State Rate ◽  
Reduced Rate ◽  
Rate Of Accumulation

Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes.

scholarly journals Immunocytochemical identification of dye-injected cells.

1982 ◽  
Vol 30 (2) ◽  
pp. 189-191 ◽  
Author(s):  
R L Michaels
Keyword(s):  
Pancreatic Islet ◽  
Islet Cells ◽  
Lucifer Yellow ◽  
Immunofluorescent Staining ◽  
Pancreatic Islet Cells ◽  
Lucifer Yellow Ch ◽  
Before And After ◽  
Coupled Cells ◽  
Indirect Immunofluorescent

Lucifer Yellow CH may be injected into pancreatic islet cells and visualized in Epon sections of the embedded tissue both before and after plastic removal and immunocyto-chemical staining. The dye retains its fluorescence, clearly marking the injected cell and adjacent dye-coupled cells, but does not interfere with the indirect immunofluorescent staining patterns that are characteristic of the islet cells

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