Reduction In Endocytosis Research Articles

Reduction of endocytosis and EGFR signaling is associated with the switch from isolated to clustered apoptosis during epithelial tissue remodeling in Drosophila.

Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Removal of cells is coordinated with the increase in number of newly dividing cells to maintain barrier function of the tissue. In Drosophila metamorphosis, larval epidermal cells (LECs) are replaced by adult precursor cells called histoblasts. Removal of LECs must counterbalance the exponentially increasing adult histoblasts. Previous work showed that the LEC removal accelerates as endocytic activity decreases throughout all LECs. Here, we show that the acceleration is accompanied by a mode switching from isolated single-cell apoptosis to clustered ones induced by the endocytic activity reduction. We identify the epidermal growth factor receptor (EGFR) pathway via extracellular-signal regulated kinase (ERK) activity as the main components downstream of endocytic activity in LECs. The reduced ERK activity, caused by the decrease in endocytic activity, is responsible for the apoptotic mode switching. Initially, ERK is transiently activated in normal LECs surrounding a single apoptotic LEC in a ligand-dependent manner, preventing clustered cell death. Following the reduction of endocytic activity, LEC apoptosis events do not provoke these transient ERK up-regulations, resulting in the acceleration of the cell elimination rate by frequent clustered apoptosis. These findings contrasted with the common perspective that clustered apoptosis is disadvantageous. Instead, switching to clustered apoptosis is required to accommodate the growth of neighboring tissues.

Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer’s Disease Mutations

We investigated early phenotypes caused by familial Alzheimer's disease (fAD) mutations in isogenic human iPSC-derived neurons. Analysis of neurons carrying fAD PS1 or APP mutations introduced using genome editing technology at the endogenous loci revealed that fAD mutant neurons had previously unreported defects in the recycling state of endocytosis and soma-to-axon transcytosis of APP and lipoproteins. The endocytosis reduction could be rescued through treatment with a β-secretase inhibitor. Our data suggest that accumulation of β-CTFs of APP, but not Aβ, slow vesicle formation from an endocytic recycling compartment marked by the transcytotic GTPase Rab11. We confirm previous results that endocytosis is affected in AD and extend these to uncover a neuron-specific defect. Decreased lipoprotein endocytosis and transcytosis to the axon suggest that a neuron-specific impairment in endocytic axonal delivery of lipoproteins and other key materials might compromise synaptic maintenance in fAD.

Regulated endosomal trafficking of Diacylglycerol lipase alpha (DAGLα) generates distinct cellular pools; implications for endocannabinoid signaling.

Diacylglycerol lipase alpha (DAGLα) generates the endocannabinoid (eCB) 2-arachidonylglycerol (2-AG) that regulates the proliferation and differentiation of neural stem cells and serves as a retrograde signaling lipid at synapses. Nothing is known about the dynamics of DAGLα expression in cells and this is important as it will govern where 2-AG can be made and released. We have developed a new construct to label DAGLα at the surface of live cells and follow its trafficking. In hippocampal neurons a cell surface pool of DAGLα co-localizes with Homer, a postsynaptic density marker. This surface pool of DAGLα is dynamic, undergoing endocytosis and recycling back to the postsynaptic membrane. A similar cycling is seen in COS-7 cells with the internalized DAGLα initially transported to EEA1 and Rab5-positive early endosomes via a clathrin-independent pathway before being transported back to the cell surface. The internalized DAGLα is present on reticular structures that co-localize with microtubules. Importantly, DAGLα cycling is a regulated process as inhibiting PKC results in a significant reduction in endocytosis. This is the first description of DAGLα cycling between the cell surface and an intracellular endosomal compartment in a manner that can regulate the level of the enzyme at the cell surface.

Bile acids reduce endocytosis of high-density lipoprotein (HDL) in HepG2 cells.

High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE) uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR) activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36). Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other.

Protein deprivation alters trafficking of the B cell receptor and endogenous antigens in B cells (APP3P.106)

Abstract Protein malnutrition in children has been linked to increased instance of infection and higher fatality likely due to limited T and B cell-mediated host immunity. These findings suggest protein malnourishment may alter antigen presentation and the cognate interaction between T and B cells. Studies here show serum deprivation induced macroauophagy and MHC class II presentation of select antigens which utilize this pathway in B cells. Further studies established serum deprivation inhibited MHC class II presentation of cytoplasmic autoantigen, glutamic acid decarboxylase and the presentation of epitopes derived from the B cell receptor. Serum deprivation did not alter the expression MHC class II proteins but did upregulate proteasome and lysosomal protease activity. Interestingly, B cell receptor accumulation on the cell surface was detected likely due to a reduction in endocytosis. An altered subcellular distribution of glutamic acid decarboxylase protein was observed, suggesting changes in the trafficking of this antigen. Studies are currently underway to examine how protein deprivation regulates antigen trafficking and presentation. This work was supported by the T32DK007519 and NIH RO1AI079065.

Investigation of endocytosis and cytotoxicity of poly-d, l-lactide-poly(ethylene glycol) micro/nano-particles in osteoblast cells

Biodegradable polymer particles present a promising approach for intracellular delivery of drugs, proteins, and nucleic acids. Poly-d,l-lactide-poly(ethylene glycol) (PELA) copolymers with different weight ratios of polyethylene glycol (PEG) were used as drug carriers in the present study. PELA particles entrapped with fluorescein isothiocyanate (FITC) as a fluorescent marker were formulated using a double emulsion-solvent evaporation technique. The size and morphology of the particles were observed with scanning electron microscope (SEM), atomic force microscope (AFM), and laser diffraction particle size analyzer (LDPSA). The purpose in the present work was to investigate the cytotoxicity and the process of endocytosis of PELA particles with different contents of PEG and variable particle size using rat osteoblasts (OBs). The cytotoxicity of the particles was investigated using 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Results indicate that as the content of PEG in the polymer increased, so did cell survival. Endocytosis was observed through a light microscope and a fluorescence microscope; intracellular uptake and retention were determined quantitatively using fluorescence spectrophotometer (FSP). The results showed that as PEG content in PELA copolymer increased, there was a reduction in endocytosis of nanoparticles in osteoblasts.

Arabidopsis phosphatidylinositol-4-monophosphate 5-kinase 4 regulates pollen tube growth and polarity by modulating membrane recycling.

Phosphatidylinositol-4-monophosphate 5-kinases produce phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] and have been implicated in vesicle trafficking and cytoskeletal rearrangements. Here, we adopted a reverse genetics approach to investigate the function of the Arabidopsis thaliana pollen-expressed gene encoding phosphatidylinositol-4-monophosphate 5-kinase 4 (PIP5K4). Pollen germination, tube growth, and polarity were significantly impaired in homozygous mutant plants lacking PIP5K4 transcript. In vitro, supplementation with PtdIns(4,5)P(2) rescued these phenotypes. In vivo, mutant pollen fertilized ovules, leading to normal seed set and silique length. However, fertilization took longer than in wild-type plants, and the pip5k4 null mutant allele was transmitted through the pollen at a reduced frequency. Analysis of endocytic events using FM1-43 (or FM4-64) suggested a reduction in endocytosis and membrane recycling in pip5k4 null mutant pollen tubes. Imaging of elongating tobacco (Nicotiana tabacum) pollen tubes transiently transformed with a PIP5K4-green fluorescent protein fusion construct revealed that the protein localized to the plasma membrane, particularly in the subapical region. Overexpression of PIP5K4-GFP delocalized the protein to the apical region of the plasma membrane, perturbed pollen tube growth, and caused apical cell wall thickening. Thus, PIP5K4 plays a crucial role in regulating the polarity of pollen tubes. This study supports a model for membrane secretion and recycling where the apical and subapical regions appear to contain the components required to promote and sustain growth.

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser(256)-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.

Long-Term Metformin Treatment Stimulates Cardiomyocyte Glucose Transport through an AMP-Activated Protein Kinase-Dependent Reduction in GLUT4 Endocytosis

Long-term (18 h) metformin treatment of cardiomyocytes increased glucose transport activity 3- to 5-fold, as measured using the phosphorylated sugar 2-deoxy-D-glucose and the nonphosphorylated sugar 3-O-methyl-D-glucose. The affinity for 3-O-methyl-D-glucose transport was not increased by metformin treatment. Total levels of glucose transporter 4 (GLUT4) were not changed by 18-h culture with or without insulin or metformin treatment. GLUT1 levels were elevated after 18 h in culture, but this increase was not altered by insulin or metformin treatment. Metformin-induced stimulation of transport was not inhibited by treatment with wortmannin and was additive with that of insulin. These data suggest that the metformin effect is mediated by a signaling route independent of phosphatidylinositol 3-kinase and Akt. Surprisingly, however, levels of both phospho-AMP-activated protein kinase and phospho-Akt were increased 4- and 3-fold, respectively, after metformin treatment. Chronic treatment with insulin for 18 h led to down-regulation of insulin-stimulated glucose transport. Cotreatment with metformin bypassed this insulin resistance by maintaining high transport levels. These data also indicate an independent point of convergence of metformin and insulin stimuli on GLUT4 regulatory processes. To test the possibility of altered GLUT4 subcellular trafficking, the kinetics of GLUT4 exocytosis and endocytosis were determined. Metformin treatment markedly slowed endocytosis of GLUT4, but exocytosis was not increased. We conclude that metformin treatment leads to a longer residence time of GLUT4 in the plasma membrane due to an AMP-activated protein kinase-dependent reduction in endocytosis. This accounts for metformin's ability to enhance hexose transport activity above insulin-stimulated and Akt-dependent levels.

The immunogenicity of dendritic cell-derived exosomes

Exosome production represents an alternate endocytic pathway for secretion. Multivesicular endosomes (MVE) fuse with the plasma membrane expelling internal vesicles or exosomes from cells. Exosome production has been recently described for immune cells including B cells, dendritic cells (DC), mast cells, macrophages and T cells. Exosomes derived from some DC populations stimulate T lymphocyte proliferation in vitro and have potent capacity to generate anti-tumour immune responses in vivo. These reported studies have involved in vitro grown mature DC expanded from precursors with cytokines. However, immature DC produce higher numbers of exosomes than mature DC and this is thought to be due to a reduction in endocytosis as DC mature, associated with reduced reformation of MVE and reduced exosome formation. This lab pioneered a method to generate immature DC in spleen long-term cultures (LTC). DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. LTC-DC produce exosomes and contain many MVE. This prompted a study of immunogenic potential with a view to the potential use of exosomes in vaccination and immunotherapy. DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. Exosomes were isolated by differential centrifugation from LTC-DC and shown by marker expression to arise by budding from the LAMP-1 + limiting endosomal membrane of MVE. These LTC-derived exosomes appear however to lack immunostimulatory markers like CD86, CD40, MHC-I and MHC-II. While LTC-DC can stimulate antigen-specific proliferation of CD4 + T cells, exosome preparations derived from antigen-pulsed DC were unable to stimulate purified naïve T cells in vitro. They were however found to weakly activate allogeneic CD8 + T cells in vitro. Tumour antigen-pulsed LTC-DC or their exosomes could induce a protective response in mice against growth of a transplanted tumour but could not induce a response to clear an existing tumour. Exosomes derived from immature DC can modulate immune responses, but do not function in direct T cell activation in vitro. Modulation of immune responses by exosomes produced by immature DC may be dependent on the presence of other antigen presenting DC subsets in the animal. The possible function of immature DC and their exosomes in maintenance of tolerance and in the induction of immunity is discussed.

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy.

Intravital two-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen within seconds. Fluorescence from a pH-insensitive conjugate of folic acid, folate Texas red (FTR), was readily observed on the apical surface of the proximal tubules and in multiple cellular compartments, but little binding or uptake could be detected in any other kidney cells. Fluorescence from a pH-sensitive conjugate of folic acid, folate fluorescein, was seen only on the apical surface of proximal tubule cells, suggesting that internalized folate conjugates are localized to acidic compartments. The majority of the FTR conjugate internalized by proximal tubules accumulated within a lysosomal pool, as determined by colocalization studies. However, portions of FTR were also shown by electron microscopy to undergo transcytosis from apical to basal domains. Additional studies with colchicine, which is known to depolymerize microtubules and interrupt transcytosis, produced a marked reduction in endocytosis of FTR, with accumulation limited to the subapical region of the cell. No evidence of cytosolic release of either folate conjugate was observed, which may represent a key difference from the cytosolic deposition seen in neoplastic cells. Together, these data support the argument that folate conjugates (and, by extrapolation, physiological folate) bind to the apical surface of proximal tubule cells and are transported into and across the cells in endocytic compartments.

Inhibition of Na+-H+ exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum.

1. Receptor-mediated endocytosis is an important mechanism for transport of macromolecules and regulation of cell-surface receptor expression. In renal proximal tubules, receptor-mediated endocytosis mediates the reabsorption of filtered albumin. Acidification of the endocytic compartments is essential because it interferes with ligand-receptor dissociation, vesicle trafficking, fusion events and coat formation. 2. Here we show that the activity of Na+-H+ exchanger isoform 3 (NHE3) is important for proper receptor-mediated endocytosis of albumin and endosomal pH homeostasis in a renal proximal tubular cell line (opossum kidney cells) which expresses NHE3 only. 3. Depending on their inhibitory potency with respect to NHE3 and their lipophilicity, the NHE inhibitors EIPA, amiloride and HOE694 differentially reduced albumin endocytosis. The hydrophilic inhibitor HOE642 had no effect. 4. Inhibition of NHE3 led to an alkalinization of early endosomes and to an acidification of the cytoplasm, indicating that Na+-H+ exchange contributes to the acidification of the early endosomal compartment due to the existence of a sufficient Na+ gradient across the endosomal membrane. 5. Exclusive acidification of the cytoplasm with propionic acid or by removal of Na+ induced a significantly smaller reduction in endocytosis than that induced by inhibition of Na+-H+ exchange. 6. Analysis of the inhibitory profiles indicates that in early endosomes and endocytic vesicles NHE3 is of major importance, whereas plasma membrane NHE3 plays a minor role. 7. Thus, NHE3-mediated acidification along the first part of the endocytic pathway plays an important role in receptor-mediated endocytosis. Furthermore, the involvement of NHE3 offers new ways to explain the regulation of receptor-mediated endocytosis.

A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters

Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.

Endocytosis is regulated by protein kinase A, but not protein kinase C in a secretory epithelial cell line

Endocytosis in the chloride secreting epithelial cell line T84 was monitored by uptake of the fluid-phase markers FITC-dextran and horseradish peroxidase (HRP). Uptake of marker was inhibited by incubation of cells at 4 degrees C, consistent with an endocytic uptake. Although activation of the cAMP-dependent second messenger pathway has been shown to stimulate exocytosis in this cell line, it caused a 63% reduction in endocytosis as measured by uptake of fluid-phase markers. In contrast, the presence of the protein kinase C activator phorbol-myristate acetate (PMA) caused no significant reduction in the level of endocytosis compared to control, nor did it reverse the inhibitory effect of PKA activation. The data thus suggest that endocytosis in T84 cells is regulated through activation of protein kinase A, but not through activation of protein kinase C.

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1279 SPECIFICITY OF RAT YOLK SAC ANTIBODIES IN PRODUCING CONGENITAL MALFORMATIONS, EMBRYONIC MALNUTRITION AND REDUCED PINOCYTOSIS

In a series of experiments first published in 1961, we reported that antibodies prepared against rat kidney were potent teratogenic agents when administered to pregnant rats (Brent 1961). It was determined that antisera which were teratogenic localized in the developing yolk sac (Slotnick and Brent, 1966). Antisera prepared against VYS proved to be the most potent teratogens, and these teratogenic antisera reduced the endocytic uptake of proteins by the VYS (Brent et al, 1972, Freedman et al, 1983). Utilizing isolectric focusing techniques, various proteins were isolated from the VYS and antisera were prepared against these individual proteins. A large number of antisera were not teratogenic and did not localize in the developing yolk sac. A smaller group of antisera were found to localize in the VYS These antisera were studied with several techniques: Determination of the endocytic index utilizing (14C) sucrose, in vivo yolk sac localization utilizing fluorescent labelled antibody and teratogenic potency. It was found that yolk sac localization of antibody specific for yolk sac antigens does not prove that the antibodies have teratogenic potency. On the other hand, if the antibodies also reduce the endocytic index, then the antibodies will invariably have teratogenic potency. Thus, the reduction of endocytosis is the first method of accurately predicting teratogenic potential without having to perform time-consuming in vivo bioassays.

Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core.

Endocytosis by cultured human skin fibroblasts of 35SO4(2-)-labelled or [3H]leucine-labelled proteoglycans from fibroblast secretions and of 125I-proteodermatan sulphate from pig skin was quantitatively investigated. The following results were obtained. (1) Core proteins prepared by digestion with chondroitin ABC lyase were at least as efficiently endocytosed as native proteoglycans. Pig skin proteodermatan sulphate was a competitive inhibitor of endocytosis of 35SO4(2-)-labelled proteoglycans. (2) Proteoglycans produced in the presence of tunicamycin and native proteoglycans degraded with endoglycosaminidase H were internalized at a normal rate. Several monosaccharides that can be bound by mammalian lectins were unable to influence the internalization of proteoglycans. Treatment of proteoglycans with neuraminidase, however, resulted in an increased clearance rate. (3) Reductive methylation or acetoacetylation of lysine residues was accompanied by a parallel decrease in the rate of proteoglycan endocytosis. Reversal of acetoacetylation normalized the uptake properties. Endocytosis of native proteoglycans was also reduced in the presence of poly-L-lysine, and this reduction in endocytosis was observed as well with proteoglycans synthesized in the presence of the lysine analogue S-2-aminoethylcysteine. These results suggest that the recognition marker required for receptor-mediated endocytosis of proteodermatan sulphate resides in its protein moiety and involves lysine residues.