scholarly journals Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p56lck, increased association with clathrin-coated pits, and altered endosomal sorting.

1993 ◽  
Vol 178 (4) ◽  
pp. 1209-1222 ◽  
Author(s):  
A Pelchen-Matthews ◽  
I J Parsons ◽  
M Marsh
Keyword(s):  
Phorbol Ester ◽  
Protein Tyrosine Kinase ◽  
Phorbol Esters ◽  
Fluid Phase ◽  
Immunofluorescent Staining ◽  
Coated Pits ◽  
Endosomal Sorting ◽  
Fluid Phase Endocytosis ◽  
Multistep Process ◽  
Nih 3T3

The phorbol ester phorbol myristate acetate (PMA) induces a rapid downregulation of CD4 from the surface of T cells and lymphocytic cell lines, as well as from CD4-transfected nonlymphoid cells. Here we have studied the mechanisms of this phorbol ester-induced CD4 modulation. Using HeLa-CD4 or NIH-3T3-CD4 cells, in which the endocytosis of CD4 is not influenced by the protein tyrosine kinase p56lck, we show that PMA enhanced the uptake of CD4, increasing the rate of CD4 endocytosis three to five-fold, and doubling the proportion of CD4 found inside the cells. Trafficking of a CD4 mutant lacking the major portion of the cytoplasmic domain, as well as fluid phase endocytosis were not affected by PMA treatment. Studies in which clathrin-coated pits were disrupted through the use of hypertonic media indicated that both the constitutive and PMA-induced CD4 uptake occurred through coated vesicles. Electron microscopy demonstrated directly that PMA increases the association of CD4 with coated pits. Immunofluorescent staining of internalized CD4 showed that PMA also diverted CD4 from the early endosome-plasma membrane recycling pathway to a mannose 6-phosphate receptor-containing late endosomal compartment. In lymphoid or p56lck-expressing transfected cells, these effects were preceded by the PMA-induced dissociation of CD4 and p56lck, which released CD4 and made possible increased endocytosis and altered intracellular trafficking. Together these results indicate that phorbol esters have multiple effects on the normal endocytosis and trafficking of CD4, and suggest that phosphorylation may influence the interaction of CD4 with coated pits.

scholarly journals The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits

1992 ◽  
Vol 117 (2) ◽  
pp. 279-290 ◽  
Author(s):  
A Pelchen-Matthews ◽  
I Boulet ◽  
DR Littman ◽  
R Fagard ◽  
M Marsh
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Protein Tyrosine ◽  
Src Gene ◽  
Fluid Phase Endocytosis ◽  
Nih 3T3

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.

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.

Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts

1999 ◽  
Vol 277 (1) ◽  
pp. C163-C173 ◽  
Author(s):  
Ester Carballo ◽  
Diana M. Pitterle ◽  
Deborah J. Stumpo ◽  
Robert T. Sperling ◽  
Perry J. Blackshear
Keyword(s):  
Phorbol Esters ◽  
Fetal Liver ◽  
Fluid Phase ◽  
Wild Type ◽  
Rhodamine Phalloidin ◽  
Kinase Substrate ◽  
Latex Beads ◽  
Fluid Phase Endocytosis ◽  
A Minor ◽  
Mannose Receptors

Macrophages express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein. To investigate a possible role of MARCKS in macrophage function, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. No differences between the wild-type and MARCKS-deficient macrophages with respect to morphology (Wright’s stain) or actin distribution (staining with rhodamine-phalloidin, under basal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed. We then evaluated phagocytosis mediated by different receptors: Fc receptors tested with IgG-coated sheep red blood cells, complement C3b receptors tested with C3b-coated yeast, mannose receptors tested with unopsonized zymosan, and nonspecific phagocytosis tested with latex beads. We also studied fluid phase endocytosis in macrophages and mouse embryo fibroblasts by using FITC-dextran to quantitate this process. In most cases, there were no differences between the cells derived from wild-type and MARCKS-deficient mice. However, a minor but significant and reproducible difference in rates of zymosan phagocytosis at 45–60 min was observed, with lower rates of phagocytosis in the MARCKS-deficient cells. Our data indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis.

scholarly journals Structure-based inhibitors reveal roles for the clathrin terminal domain and its W-box binding site in CME

2020 ◽  
Author(s):  
Zhiming Chen ◽  
Rosa Mino ◽  
Marcel Mettlen ◽  
Peter Michaely ◽  
Madhura Bhave ◽  
...  
Keyword(s):  
Binding Sites ◽  
Fluid Phase ◽  
Structure Design ◽  
Coated Pits ◽  
Bulk Fluid ◽  
Lysosomal Trafficking ◽  
Terminal Domain ◽  
Binding Surface ◽  
Fluid Phase Endocytosis ◽  
Late Stages

AbstractClathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain (TD), which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the TD with or without the distal leg specifically inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. We designed small membrane-penetrating peptides that mimic the four known binding sites on the TD. A peptide, Wbox2, designed to mimic to the W-box motif binding surface on TD binds to SNX9 and AP2, and potently and acutely inhibits CME, while not perturbing AP1-dependent lysosomal trafficking from the Golgi or bulk, fluid phase endocytosis.SummaryChen et al define the role the N-terminal domain (TD) of clathrin heavy chain in early and late stages of clathrin-mediated endocytosis, and guided by its structure, design a membrane-penetrating peptide, Wbox2, that acutely and potently inhibits CME.

Regulation of interacting populations during endocytosis: models of growth factor-tumor promoter dynamics

1986 ◽  
Vol 250 (6) ◽  
pp. R1123-R1132
Author(s):  
M. Gex-Fabry ◽  
C. DeLisi
Keyword(s):  
Growth Factor ◽  
Ligand Binding ◽  
Phorbol Ester ◽  
Egf Receptor ◽  
Phorbol Esters ◽  
Time Lag ◽  
Tumor Promoter ◽  
Necessary Condition ◽  
Coated Pits ◽  
Receptor Complexes

A model of growth factor-cell receptor interactions, including internalization, sorting, recycling, and degradation and their modulation by tumor promoters, is developed, analyzed, and tested. In keeping with data and concepts based on a large number of systems, the main assumption is that after receptor-ligand binding the complex associates with a second membrane protein, localized in coated pits, and that this event is a necessary condition for receptor-mediated endocytosis and subsequent intracellular processes. As a consequence of the model, ligands having distinct receptors interfere at the cell surface through competition between their receptor complexes for a limited pool of coated pit proteins. The utility of the model is illustrated by a detailed analysis of binding, endocytosis, and degradation of epidermal growth factor (EGF) and their modulation by phorbol esters. The analysis permits quantitative characterization of the dynamics of the endocytic processes and leads to the following conclusions. The Scatchard plot changes from linear to nonlinear as the ratio of the number of coated pit proteins to the number of receptors decreases. Competition between phorbol ester and EGF-bound receptors for coated pit proteins predicts, in agreement with observation, conversion of nonlinear EGF Scatchard plots to linear plots subsequent to reincubation with phorbol esters. The postulated competition suggests a local homology between the phorbol ester receptor and the EGF receptor. Homologous and heterologous downregulations observed in numerous systems are natural consequences of the model. Preincubation with the heterologous ligand increases the time lag between ligand binding and lysosomal degradation and alters intracellular sorting.

Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion

1999 ◽  
Vol 112 (15) ◽  
pp. 2549-2557 ◽  
Author(s):  
A. Aballay ◽  
P.D. Stahl ◽  
L.S. Mayorga
Keyword(s):  
Phorbol Ester ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Early Endosomes ◽  
Zinc Depletion ◽  
Fluid Phase Endocytosis ◽  
Endocytic Vesicles

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.

scholarly journals Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells.

1991 ◽  
Vol 173 (3) ◽  
pp. 575-587 ◽  
Author(s):  
A Pelchen-Matthews ◽  
J E Armes ◽  
G Griffiths ◽  
M Marsh
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Hela Cells ◽  
Protein Tyrosine Kinase ◽  
Fluid Phase ◽  
Cytoplasmic Domain ◽  
Lymphoid Cells ◽  
Surface Proteins ◽  
Coated Pits ◽  
T Cell Lines

The endocytosis of the T cell differentiation antigen CD4 has been investigated in CD4-transfected HeLa cells, the promyelocytic HL-60 cell line, and in a number of leukemia- or lymphoma-derived T cell lines. CD4 internalization was followed using radioiodinated antibodies in an acid-elution endocytosis assay, or by covalently modifying cell surface proteins with biotin and analyzing CD4 distributions by immunoprecipitation; both approaches gave equivalent results. The assays demonstrated that in transfected HeLa cells and in HL-60 cells CD4 was constitutively internalized and recycled in the absence of ligand. Immunogold labeling and electron microscopy demonstrated that CD4 enters cells through coated pits. In contrast to the nonlymphocytic cells, T cell lines showed very little endocytosis of CD4. Measurements of fluid phase endocytosis and morphometric analysis of the endosome compartment indicated that the endocytic capacities of HeLa and lymphoid cells are equivalent and suggested that the low level of CD4 uptake in lymphocytic cells is due to exclusion of CD4 from coated pits. This conclusion was supported by experiments using truncated CD4 molecules, lacking the bulk of the cytoplasmic domain, which were internalized equally efficiently in both transfected lymphocytes and HeLa cells. Together, these results indicate that the cytoplasmic domain of CD4 mediates the different interactions with the endocytic apparatus in lymphoid and nonlymphoid cells. We suggest that the CD4-associated lymphocyte-specific protein tyrosine kinase p56lck may be involved in preventing CD4 endocytosis in T cells.

scholarly journals Cell surface dynamics of neutrophils stimulated with phorbol esters or retinoids.

1987 ◽  
Vol 105 (1) ◽  
pp. 417-426 ◽  
Author(s):  
J M Robinson ◽  
J A Badwey ◽  
M L Karnovsky ◽  
M J Karnovsky
Keyword(s):  
Phorbol Esters ◽  
Morphological Changes ◽  
Fluid Phase ◽  
Dependent Manner ◽  
Surface Dynamics ◽  
Cytochemical Localization ◽  
Fluid Phase Endocytosis ◽  
Kinetics Of ◽  
Dose Dependent ◽  
Superoxide Release

Neutrophils undergo rapid morphological changes as well as metabolic perturbations when stimulated with certain phorbol esters. Stimulated cells initially exhibit pronounced projections emanating from the cell bodies, followed by rounding of the cells, reduction in granule number, and the appearance of intracellular vesicles. We show these vesicles to be derived, at least in part, from the plasmalemma. The experimental approach involved labeling stimulated and unstimulated cells with native ferritin and cationized ferritin, along with the cytochemical localization of ecto-5'-nucleotidase. The labeling patterns of the vesicles indicate that these structures are involved in both phorbol ester-stimulated adsorptive and fluid-phase endocytosis. Neutrophils stimulated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibit two distinct rates of superoxide release in which the second, prolonged level is approximately 50% of the initial rate. All-trans-retinal, which we have recently shown to stimulate O2- release but not granule exocytosis or cell vesiculation, induces a single prolonged rate of maximal O2- release. Neutrophils treated with both all-trans-retinal and TPA exhibit only a single sustained rate of maximal O2- release similar to that observed with all-trans-retinal alone. Moreover, treatment of cells with all-trans-retinal blocks the vesiculation of neutrophils induced by TPA in a dose-dependent manner. This observation provides a possible explanation for the differences in the kinetics of superoxide release.

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.

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.

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