Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy

1993 ◽  
Vol 105 (3) ◽  
pp. 861-866 ◽  
Author(s):  
L. Aubry ◽  
G. Klein ◽  
J.L. Martiel ◽  
M. Satre
Keyword(s):  
Dictyostelium Discoideum ◽  
Fluid Phase ◽  
Lag Phase ◽  
Nutritive Medium ◽  
Chase Period ◽  
Endosomal Acidification ◽  
Fluid Phase Endocytosis ◽  
Endosomal Ph ◽  
Efflux Kinetics ◽  
Secondary Lysosomes

The evolution of endo-lysosomal pH in Dictyostelium discoideum amoebae was examined during fluid-phase endocytosis. Pulse-chase experiments were conducted in nutritive medium or in non-nutritive medium using fluorescein labelled dextran (FITC-dextran) as fluid-phase marker and pH probe. In both conditions, efflux kinetics were characterized by an extended lag phase lasting for 45–60 min and corresponding to intracellular transit of FITC-dextran cohort. During the chase period, endosomal pH decreased during approximately 20 min from extracellular pH down to pH 4.6-5.0, then, it increased within the next 20–40 min to reach pH 6.0-6.2. It was only at this stage that FITC-dextran was released back into the medium with pseudo first-order kinetics. A vacuolar H(+)-ATPase is involved in endosomal acidification as the acidification process was markedly reduced in mutant strain HGR8, partially defective in vacuolar H(+)-ATPase and in parent type strain AX2 by bafilomycin A1, a selective inhibitor of this enzyme. Our data suggest that endocytic cargo is channeled from endosomes to secondary lysosomes that are actively linked to the plasma membrane via recycling vesicles.

scholarly journals A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis.

1989 ◽  
Vol 109 (4) ◽  
pp. 1445-1456 ◽  
Author(s):  
D L Ebert ◽  
H H Freeze ◽  
J Richardson ◽  
R L Dimond ◽  
J A Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Mutant Strain ◽  
Lysosomal Enzyme ◽  
Fluid Phase ◽  
Wild Type ◽  
Lysosomal Hydrolases ◽  
Chase Period ◽  
Secondary Lysosomes ◽  
Beta Glucosidase ◽  
Enzyme Targeting

A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha-mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild-type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the alpha-mannosidase precursor by HMW570 was not accompanied by major alterations in N-linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominantly located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.

Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization

1999 ◽  
Vol 112 (19) ◽  
pp. 3195-3203 ◽  
Author(s):  
A.A. Noegel ◽  
F. Rivero ◽  
R. Albrecht ◽  
K.P. Janssen ◽  
J. Kohler ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Fluid Phase ◽  
Axenic Medium ◽  
Terminal Domain ◽  
Defect Growth ◽  
Fluid Phase Endocytosis ◽  
Altered Cell ◽  
Mutant Cells ◽  
Proline Rich Region

The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.

Endosomal alkalinization reduces Jmax and Km of albumin receptor-mediated endocytosis in OK cells

1995 ◽  
Vol 268 (5) ◽  
pp. F899-F906 ◽  
Author(s):  
M. Gekle ◽  
S. Mildenberger ◽  
R. Freudinger ◽  
S. Silbernagl
Keyword(s):  
Proximal Tubule ◽  
Prolonged Exposure ◽  
Specific Binding ◽  
Fluid Phase ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1 ◽  
Receptor Mediated Endocytosis ◽  
Fluid Phase Endocytosis ◽  
Endosomal Ph ◽  
Kinetics Of

In this study, we investigated the effects of endosomal alkalinization on kinetics of endocytotic uptake in intact proximal tubule-derived opossum kidney cells. We used fluorescein isothiocyanate (FITC)-labeled albumin and FITC-dextran as endocytotic substrates for receptor-mediated endocytosis and fluid-phase endocytosis, respectively. The pH in endosomes labeled with either FITC-albumin or FITC-dextran rose in the presence of the vacuolar-type ATPase inhibitor, bafilomycin A1, and in the presence of NH4Cl. Cytoplasmic pH, decreased in the presence of bafilomycin A1, but was not significantly different from control during prolonged exposure of the cells to NH4Cl. Endocytotic uptake of FITC-dextran was not affected by endosomal pH changes. Endocytotic uptake of FITC-albumin was reduced markedly by bafilomycin A1 (decrease of maximum transport rate and apparent affinity). Selective alkalinization of endosomes using NH4Cl (i.e., with the cytoplasmic pH not different from control) reduced FITC-albumin uptake in a similar way but to a lesser extent than did bafilomycin A1. Intracellular albumin degradation was impaired by bafilomycin A1 and NH4Cl. Prevention of endosome-lysosome fusion (lowering the temperature to 20 degrees C) abolished the effects of endosomal alkalinization. Furthermore, specific binding of albumin to the plasma membrane was reduced after incubation with bafilomycin A1, indicating an impairment of receptor recycling. These data show that endosomal pH is an important determinant for the kinetics of receptor-mediated endocytotic uptake of albumin in the proximal tubule but not for fluid-phase endocytosis. Endosomal alkalinization disturbs intracellular ligand handling and receptor trafficking, leading to a reduction of endocytotic capacity and affinity.

scholarly journals Dictyostelium discoideum mutants with conditional defects in phagocytosis.

1994 ◽  
Vol 126 (4) ◽  
pp. 955-966 ◽  
Author(s):  
C J Cohen ◽  
R Bacon ◽  
M Clarke ◽  
K Joiner ◽  
I Mellman
Keyword(s):  
Dictyostelium Discoideum ◽  
Single Gene ◽  
Fluid Phase ◽  
Polystyrene Latex ◽  
Expression Cloning ◽  
Latex Beads ◽  
Solid Substrates ◽  
Fluid Phase Endocytosis ◽  
Plasma Membrane Receptor ◽  
Mutant Phenotypes

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s).

Cytosolic entry of Bisphosphonates into macrophages and osteoclasts requires fluid-phase endocytosis and endosomal acidification

Bone ◽  
2006 ◽  
Vol 38 (3) ◽  
pp. 47
Author(s):  
Keith Thompson ◽  
Michael J. Rogers ◽  
Fraser P. Coxon ◽  
Julie C. Crockett
Keyword(s):  
Fluid Phase ◽  
Endosomal Acidification ◽  
Fluid Phase Endocytosis

scholarly journals Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver.

1990 ◽  
Vol 38 (4) ◽  
pp. 515-524 ◽  
Author(s):  
M Ishii ◽  
B Vroman ◽  
N F LaRusso
Keyword(s):  
Acid Phosphatase ◽  
Bile Duct ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Rat Liver ◽  
Intrahepatic Bile Duct ◽  
Fluid Phase ◽  
Fluid Phase Endocytosis ◽  
Normal Rat ◽  
Secondary Lysosomes

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.

The effects of osmotic pressure changes on the germination of Dictyostelium discoideum spores

1977 ◽  
Vol 23 (9) ◽  
pp. 1170-1177 ◽  
Author(s):  
David A. Cotter
Keyword(s):  
Ethylene Glycol ◽  
Dictyostelium Discoideum ◽  
Room Temperature ◽  
Thermal Inactivation ◽  
Sucrose Concentration ◽  
Lag Phase ◽  
Activation Process ◽  
Sucrose Solutions ◽  
Spore Population ◽  
Pressure Changes

Polyalcohols such as ethylene glycol and glycerol at 3 M penetrate and activate spores of Dictyostelium discoideum incubated at room temperature. Higher concentrations of ethylene glycol result in lysis upon suspension of spores in dilute phosphate buffer. Erythritol and arabitol at 3 M do not penetrate or activate D. discoideum spores.Air-dried spores or those incubated in 2 M sucrose solutions are not activated with the usual heat treatment of 45 °C for 30 min. The plasmolyzed spores are activated at temperatures above 45 °C when heated in the presence of 2 M sucrose for 30 min. The temperature for maximum activation and the temperature for thermal inactivation of spores are raised 7–10 °C in high sucrose concentrations. Long-term incubation of heat-activated spores in 2 M sucrose solutions does not result in a return to dormancy.Moderate sucrose concentrations near 0.2 M do not block the heat-induced activation process but must be removed from the spore population to prevent a return to dormancy within 6 h. Other polyhydric compounds at 0.25 M concentration also cause spore deactivation within 6 h of room temperature incubation. Oxygen uptake of spores undergoing deactivation in 0.18 M sucrose is inhibited as compared to control levels. Moderate concentrations of sucrose do not block the early events of postactivation lag and the spores accumulate at the end of the lag phase. The longer the spores remain unswollen at the end of the postactivation lag phase, the greater the percentage of spores which return to dormancy. The effects of moderate sucrose concentration (lowered water activity) are not duplicated by the same quantity of Ficoll, indicating that the colligative properties of the sucrose solutions are responsible for deactivation.

Synaptojanin family members are implicated in endocytic membrane traffic in yeast

1998 ◽  
Vol 111 (22) ◽  
pp. 3347-3356 ◽  
Author(s):  
B. Singer-Kruger ◽  
Y. Nemoto ◽  
L. Daniell ◽  
S. Ferro-Novick ◽  
P. De Camilli
Keyword(s):  
Synaptic Vesicles ◽  
Direct Evidence ◽  
Family Members ◽  
Fluid Phase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Close Link ◽  
Fluid Phase Endocytosis ◽  
Synaptojanin 1

The synaptojanins represent a subfamily of inositol 5′-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.

scholarly journals Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway

2020 ◽  
Author(s):  
Chaitra Prabhakara ◽  
Rashmi Godbole ◽  
Parijat Sil ◽  
Sowmya Jahnavi ◽  
Thomas S van Zanten ◽  
...  
Keyword(s):  
Viral Infection ◽  
Receptor Binding ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Entry Inhibitor ◽  
Receptor Binding Domain ◽  
Endosomal Acidification ◽  
Independent Mechanism ◽  
Ph Dependent ◽  
Endosomal Ph

AbstractMany viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.

scholarly journals Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

2021 ◽  
Author(s):  
Farnaz Fekri ◽  
Ralph Christian Delos Santos ◽  
Raffi Karshafian ◽  
Costin N. Antonescu
Keyword(s):  
Drug Delivery ◽  
Fluid Phase ◽  
Acid Sphingomyelinase ◽  
Coated Pits ◽  
Drug Permeability ◽  
Membrane Pores ◽  
Signaling Mechanisms ◽  
Sheer Stress ◽  
Cell Plasma ◽  
Fluid Phase Endocytosis

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

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