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

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.

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Synaptojanin family members are implicated in endocytic membrane traffic in yeast

Journal of Cell Science ◽

10.1242/jcs.111.22.3347 ◽

1998 ◽

Vol 111 (22) ◽

pp. 3347-3356 ◽

Cited By ~ 2

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.

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

10.32920/14638422 ◽

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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Effect of serum proteins on sodium-linked transport processes in cultured kidney cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v5111964 ◽

1995 ◽

Vol 5 (11) ◽

pp. 1964-1970

Author(s):

S S Blumenthal ◽

D L Lewand ◽

P A Tipnis ◽

J G Kleinman

Keyword(s):

Amino Acid ◽

Dextran Sulfate ◽

Cultured Cells ◽

Serum Proteins ◽

Fluid Phase ◽

Defined Medium ◽

Transport Processes ◽

Transport Systems ◽

Heat Treated ◽

Fluid Phase Endocytosis

The mechanism for increased Na+ retention in the nephrotic syndrome is unknown. To determine if Na+ transport systems in the proximal tubule might be affected by filtered proteins, mouse cortical tubule cells grown in defined medium were exposed to concentrations of bovine serum albumin (BSA) ranging from 0.01 to 0.5%. Activity of the Na(+)-glucose cotransporter, measured as Na(+)-dependent uptake of alpha-methylglucoside, increased progressively to a maximum of 2.3-fold above baseline (P < 0.001; N = 10). The increase in transporter activity was due to an increased Vmax, and the magnitude of the increase was inversely related to the basal cotransporter activity of the cultures. Increased cotransporter activity was detectable 6 h after exposure, was sustained for 24 h after cells were removed from an albumin-free medium, and was prevented by cycloheximide. Heat-treated BSA, fatty-acid and globulin-free BSA, and gamma-globulins were as effective at increasing Na(+)-glucose cotransporter activity as untreated Fraction V BSA. Dextran, dextran-sulfate, and amino acid supplements were ineffective. Neither protease inhibitors nor chloroquine added to an albumin-containing medium prevented increased alpha-methylglucoside uptake. Albumin did not change the rate of fluid-phase endocytosis in the cultured cells. Na(+)-amino acid cotransport and Na(+)-H+ exchange were either decreased or unchanged after BSA exposure. Exposing apical surfaces of cells grown on permeable membranes to BSA led to a greater increase in activity of the Na(+)-glucose cotransporter relative to controls than did exposing the basolateral surface (145 versus 89%; P < 0.05; N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

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Fluid phase endocytosis by isolated rabbit lacrimal gland acinar cells

Experimental Eye Research ◽

10.1016/s0014-4835(05)80066-1 ◽

1995 ◽

Vol 60 (5) ◽

pp. 511-525 ◽

Cited By ~ 38

Author(s):

J. Peter Gierow ◽

Robert W. Lambert ◽

Austin K. Mircheff

Keyword(s):

Lacrimal Gland ◽

Acinar Cells ◽

Fluid Phase ◽

Fluid Phase Endocytosis

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[39] Assay of growth factor stimulation of fluid-phase endocytosis

Peptide Growth Factors - Part A - Methods in Enzymology ◽

10.1016/s0076-6879(87)46042-4 ◽

1987 ◽

pp. 402-417 ◽

Cited By ~ 6

Author(s):

H. Steven Wiley ◽

Dana N. McKinley

Keyword(s):

Growth Factor ◽

Fluid Phase ◽

Fluid Phase Endocytosis ◽

Stimulation Of

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Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes

Journal of Cell Science ◽

10.1242/jcs.108.9.2983 ◽

1995 ◽

Vol 108 (9) ◽

pp. 2983-2991 ◽

Cited By ~ 5

Author(s):

R.H. Moore ◽

N. Sadovnikoff ◽

S. Hoffenberg ◽

S. Liu ◽

P. Woodford ◽

...

Keyword(s):

Transferrin Receptor ◽

Adrenergic Receptor ◽

Fluid Phase ◽

Small Gtpase ◽

Endocytic Pathway ◽

Receptor Internalization ◽

Beta Agonist ◽

Hek293 Cells ◽

Fluid Phase Endocytosis ◽

Beta 2

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.

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The Dictyostelium RasS protein is required for macropinocytosis, phagocytosis and the control of cell movement

Journal of Cell Science ◽

10.1242/jcs.113.4.709 ◽

2000 ◽

Vol 113 (4) ◽

pp. 709-719 ◽

Cited By ~ 4

Author(s):

J.R. Chubb ◽

A. Wilkins ◽

G.M. Thomas ◽

R.H. Insall

Keyword(s):

Cell Migration ◽

Significant Proportion ◽

Cell Movement ◽

Fluid Phase ◽

Small Gtpases ◽

Cell Cortex ◽

Fluid Phase Endocytosis ◽

Ras Family ◽

Actin Protrusions ◽

Mutant Cells

Endocytosis and cell migration both require transient localised remodelling of the cell cortex. Several lines of evidence suggest a key regulatory role in these activities for members of the Ras family of small GTPases. We have generated Dictyostelium cells lacking one member of this family, RasS, and the mutant cells are perturbed in endocytosis and cell migration. Mutant amoebae are defective in phagocytosis and fluid-phase endocytosis and are impaired in growth. Conversely, the rasS(-)cells show an enhanced rate of cell migration, moving three times faster than wild-type controls. The mutant cells display an aberrant morphology, are highly polarised, carry many elongated actin protrusions and show a concomitant decrease in formation of pinocytic crowns on the cell surface. These morphological aberrations are paralleled by changes in the actin cytoskeleton, with a significant proportion of the cortical F-actin relocalised to prominent pseudopodia. Rapid migration and endocytosis appear to be mutually incompatible and it is likely that RasS protein is required to maintain the normal balance between these two actin-dependent processes.

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Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins

Biochemical Journal ◽

10.1042/bj2810809 ◽

1992 ◽

Vol 281 (3) ◽

pp. 809-817 ◽

Cited By ~ 32

Author(s):

J Yang ◽

A E Clark ◽

R Harrison ◽

I J Kozka ◽

G D Holman

Keyword(s):

Cell Surface ◽

Glucose Transport ◽

Glucose Transporter ◽

Fluid Phase ◽

Transport Activity ◽

Phenylarsine Oxide ◽

Fluid Phase Endocytosis ◽

Surface Labelling ◽

Slow Dissociation ◽

Stimulation Of

We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled.

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