Fluid-phase Endocytosis Research Articles

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.

Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation

AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced AS1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that cancer cells may overexpress nucleolin to surmount this barrier.

Characterization of Atp6ap2 ((Pro)‐Renin Receptor) in Receptor Mediated Endocytosis by the Renal Proximal Tubule

Atp6ap2 may be an accessory subunit of the V‐type H+ ATPase regulating organellar, cellular, and systemic acid‐base homeostasis. Here we examine whether the knock‐down of Atp6ap2 affects renal V‐ATPase function in the proximal tubule. There, V‐ATPases are important for receptor‐mediated endocytosis of low molecular weight proteins from urine via the megalin/cubilin receptors. In order to elucidate the role of Atp6ap2 in this function we used an inducible shRNA Atp6ap2 rat model and a doxycyline‐inducible kidney specific Atp6ap2 KO mouse model. Knock‐down of Atp6ap2 reduced Atp6ap2 expression by approximately 90 %. Atp6ap2 knock‐down animals showed higher proteinuria (albumin and Vitamin D binding protein).To further examine proximal tubular endocytosis, we injected animals with markers for fluid phase endocytosis (FITC‐ dextran, 10 kDa) and a for receptor mediated endocytosis (human transferrin). Immunofluorescence of kidney slices from both animal groups did not show a difference in FITC‐dextran intensity or localization. Furthermore, evaluation of receptor mediated endocytosis of human transferrin, showed in immunofluorescence stainings no differences between the animal groups but the knock‐down animals excreted human transferrin in the urine while control animal did not so. Hence, our data suggests a possible role for ATP6ap2 for proximal tubule function in the kidney; however, the defect in receptor‐mediated endocytosis is mild in rats and mice.Funded by the Swiss National Science Foundation.

Non-apoptotic cell death associated with perturbations of macropinocytosis.

Although macropinocytosis is widely recognized as a distinct form of fluid-phase endocytosis in antigen-presenting dendritic cells, it also occurs constitutively in many other normal and transformed cell types. Recent studies have established that various genetic or pharmacological manipulations can hyperstimulate macropinocytosis or disrupt normal macropinosome trafficking pathways, leading to accumulation of greatly enlarged cytoplasmic vacuoles. In some cases, this extreme vacuolization is associated with a unique form of non-apoptotic cell death termed “methuosis,” from the Greek methuo (to drink to intoxication). It remains unclear whether cell death related to dysfunctional macropinocytosis occurs in normal physiological contexts. However, the finding that some types of cancer cells are particularly vulnerable to this unusual form of cell death has raised the possibility that small molecules capable of altering macropinosome trafficking or function might be useful as therapeutic agents against cancers that are resistant to drugs that work by inducing apoptosis. Herein we review examples of cell death associated with dysfunctional macropinocytosis and summarize what is known about the underlying mechanisms.

The effect of the size of fluorescent dextran on its endocytic pathway.

Fluorescent dextrans are commonly used as macropinocytic probes to study the properties of endocytic cargoes; however, the effect of the size of dextrans on endocytic mechanisms has not been carefully analyzed. By using chemical and siRNA inhibition of individual endocytic pathways, we evaluated the internalization of two commonly used dextrans, Dex10 (dextran 10 kDa) and Dex70 (dextran 70 kDa), in mammalian HeLa cells and Caenorhabditis elegans coelomocytes. We revealed that Dex70 enters these two cell types predominantly via clathrin- and dynamin-independent and amiloride-sensitive macropinocytosis process; Dex10, on the other hand, enters the two cell types through clathrin-/dynamin-dependent micropinocytosis in addition to macropinocytosis. In addition, although different-sized dextrans follow different endocytic processes, they share common post-endocytic events. Herein, though straightforward, our studies support that the size of nanomaterials could play a paramount role in their inclusion into endocytic vesicles and suggest that care should be taken while selecting endocytic pathway markers. Based on our results, we propose that Dex70 is a better probe for macropinocytosis, whereas Dex10 and smaller molecules are better for probing general fluid-phase endocytosis, which includes macropinocytic and micropinocytic processes.

TOM1 is a PI5P effector involved in the regulation of endosomal maturation.

Phosphoinositides represent a major class of lipids specifically involved in the organization of signaling cascades, maintenance of the identity of organelles and regulation of multiple intracellular trafficking steps. We previously reported that phosphatidylinositol 5-monophosphate (PI5P), produced by the Shigella flexneri phosphatase IpgD, is implicated in the endosomal sorting of the epidermal growth factor receptor (EGFR). Here, we show that the adaptor protein TOM1 is a new direct binding partner of PI5P. We identify the domain of TOM1 involved in this interaction and characterize the binding motif. Finally, we demonstrate that the recruitment of TOM1 by PI5P on signaling endosomes is responsible for the delay in EGFR degradation and fluid-phase bulk endocytosis. Taken together, our data strongly suggest that PI5P enrichment in signaling endosomes prevents endosomal maturation through the recruitment of TOM1, and point to a new function of PI5P in regulating discrete maturation steps in the endosomal system.

Identification and characterization of novel protein-derived arginine-rich cell-penetrating peptides

Cell-penetrating peptides (CPPs) have proven their potential as an efficient delivery system due to their intrinsic ability to traverse biological membranes and transport various cargoes into the cells. In the present study, we have identified novel natural protein-derived CPPs using an integrated (in silico and experimental) approach. First, using bioinformatics approach, arginine-rich peptide segments were extracted from SwissProt proteins and their cell-penetrating properties were predicted. Finally, eight peptides were selected and their internalization was validated using experimental techniques. Laser scanning confocal microscopy and flow cytometry confirmed that seven out of eight peptides were internalized into live cells with varying efficiencies without significant cytotoxicity. Three peptides have shown higher internalization efficacy than TAT peptide, the most widely used CPP. Among these three peptides, one peptide (P8), derived from voltage-dependent L-type calcium channel subunit alpha-1D, was able to accumulate inside in a variety of cell types very efficiently through a rapid dose-dependent process. Further, experiments involving inhibition with various endocytic inhibitors along with co-localization studies indicate that the uptake mechanism of P8 is macropinocytosis, a fluid-phase endocytosis process. In addition, competitive inhibition with heparin revealed the involvement of cell-surface proteoglycans in P8 uptake. In summary, the present study provides evidence that an integrated in silico and experimental approach is an effective strategy for the identification of novel CPPs and CPPs identified in the present study have promising perspectives for future drug delivery applications.

Reduced Intracellular Drug Accumulation in Drug-Resistant Leukemia Cells is Not Only Solely Due to MDR-Mediated Efflux but also to Decreased Uptake.

Expression of ABC family transporter proteins that promote drug efflux from cancer cells is a widely observed mechanism of multi-drug resistance of cancer cells. Cell adaptation in long-term culture of HL60 leukemic cells in the presence of chemotherapy leads to induction and maintenance of the ABC transporters expression, preventing further accumulation of drugs. However, we found that decreased accumulation of drugs and fluorescent dyes also contributed by a reduced uptake by the resistant cells. Confocal time-lapse microscopy and flow cytometry revealed that fluid-phase endocytosis was diminished in drug-resistant cells compared to drug-sensitive cells. Drug uptake was increased by insulin co-treatment when cells were grown in methylcellulose and monitored under the microscope, but not when cultured in suspension. We propose that multi-drug resistance is not only solely achieved by enhanced efflux capacity but also by supressed intake of the drug, offering an alternative target to overcome drug resistance or potentiate chemotherapy.

Distinct Biochemical and Functional Properties of Two Rab5 Homologs from the Rice Blast Fungus Magnaporthe oryzae

Rab5 is a key regulator of early endocytosis by promoting early endosomal fusion and motility. In this study, we have unexpectedly found distinct properties of the two Rab5 homologs (MoRab5A and MoRab5B) from Magnaporthe oryzae, a pathogenic fungus in plants whose infection causes rice blast disease. Like mammalian Rab5, MoRab5A and MoRab5B can bind to several Rab5 effectors in a GTP-dependent manner, including EEA1, Rabenosyn-5, and Rabaptin-5. However, MoRab5A shows distinct binding characteristics in the sense that both the wild-type and the GTP hydrolysis-defective constitutively active mutant bind the effectors equally well in GST pull-down assays, suggesting that MoRab5A is defective in GTP hydrolysis and mostly in the GTP-bound conformation in the cell. Indeed, GTP hydrolysis assays indicate that MoRab5A GTPase activity is dramatically lower than MoRab5B and human Rab5 and is insensitive to RabGAP5 stimulation. We have further identified a Pro residue in the switch I region largely responsible for the distinct MoRab5A properties by characterization of MoRab5A and MoRab5B chimeras and mutagenesis. The differences between MoRab5A and MoRab5B extend to their functions in the cell. Although they both target to early endosomes, only MoRab5B closely resembles human Rab5 in promoting early endosome fusion and stimulating fluid phase endocytosis. In contrast, MoRab5A correlates with another related early endosomal Rab, Rab22, in terms of the presence of the switch I Pro residue and the blocked GTPase activity. Our data thus identify MoRab5B as the Rab5 ortholog and suggest that MoRab5A specializes to perform a non-redundant function in endosomal sorting.

Abstract 5374: Effect of carbon nanotube amount and subcellular location on the near infrared (NIR) photothermal ablation of cells

Abstract Background and Objective: Single-walled carbon nanotubes (SWNTs) are readily taken up by cells, strongly absorb in the NIR (700-1100 nm) region, and efficiently convert absorbed NIR into heat. These properties have made SWNTs an interesting facilitator for the NIR mediated thermal ablation of cancer cells. In the application of SWNTs to thermal ablation, it is not known what amount of cell-associated SWNTs is required to achieve a defined level of cell killing upon NIR irradiation. In addition, it has not been quantitatively demonstrated whether SWNTs bound to the plasma membrane or internalized into vesicles are more efficient in killing cells. The overall objective of this project is to assess NIR mediated killing as a function of cell-associated SWNT amount and SWNT subcellular location. Methods: The study was performed in three phases. The first phase involved non-covalent functionalization of SWNTs by bovine serum albumin. Non-targeted uptake and NIR irradiation of cells was performed to find the optimal laser power density, NIR exposure time and SWNT concentration to kill cells. SWNTs associated with cells were then extracted and quantified by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique (Wang et al., 2009, Anal. Chem. 81:2944-2952) and the SWNT amount was correlated with cell killing. The second phase included the non-covalent functionalization of SWNTs with phospholipid-PEG conjugated folic acid (PL-PEG-FA) as a targeting ligand. The SWNTs were internalized by receptor-mediated endocytosis at 37°C, the optimum laser conditions were determined as in the first phase, and the amount of internalized SWNTs were quantified and correlated with NIR-mediated cell killing. In the third phase, cells were incubated with the PL-PEG-FA SWNTs at 4°C to allow binding to the cell surface without internalization, followed by measuring the amount of surface-bound SWNTs. The amount of surface-bound SWNTs was then correlated with NIR-mediated cell death. Results: An increase in killing of cells incubated with SWNTs was observed in both the non-targeted and targeted systems as a function of NIR laser power density, NIR exposure time and incubated SWNT concentration. With the folate targeted system, there was more cell death when SWNTs were internalized into vesicles compared to when an equivalent amount of SWNTs were confined to the cell surface. Conclusion: SWNTs internalized by cells, either by fluid phase endocytosis, or by receptor-mediated endocytosis via the folate receptor, were effective in killing cells upon NIR irradiation. However, SWNTs confined to the cell surface after binding to folate receptors at low temperature were not as efficient in killing cells upon NIR irradiation. This suggests that NIR-mediated thermal ablation therapy will be more effective when SWNTs are targeted to a receptor and are internalized by receptor-mediated endocytosis. Citation Format: Vasanth Siruvallur Murali, Carole Mikoryak, Ruhung Wang, Rockford K. Draper. Effect of carbon nanotube amount and subcellular location on the near infrared (NIR) photothermal ablation of cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5374. doi:10.1158/1538-7445.AM2014-5374

The involvement of selected membrane transport mechanisms in the cellular uptake of 177Lu-labeled bombesin, somatostatin and gastrin analogues

IntroductionRadiolabeled receptor-targeting peptides are a useful tool for the diagnostic imaging and radiotherapy of some malignancies. However, the retention of radioactivity in the kidney may result in renal radiotoxic injury. This study seeks to evaluate the role of endocytic receptor megalin, renal SLC influx transporters and fluid phase endocytosis (FPE) in the cellular accumulation of radiolabeled peptides. MethodsIn vitro transport cellular studies using megalin ligands (RAP, albumin), fluid phase endocytosis (FPE) inhibitor rottlerin and low temperature were employed to evaluate the transport mechanisms of the peptides. Cells transfected with hOAT1 or hOCT2 were used to analyze the role of these SLC transporters. Somatostatin (177Lu-DOTA-[Tyr3]octreotate, 177Lu-DOTA-[1-Nal3]octreotide), gastrin (177Lu-DOTA-sargastrin) and bombesin (177Lu-DOTA-[Pro1,Tyr4]bombesin, 177Lu-DOTA-[Lys3]bombesin, 177Lu-PCTA-[Lys3]bombesin) analogues were involved in the study. ResultsRAP, albumin and low temperature decreased the accumulation of all the studied peptides significantly. With one exception, rottlerin caused the concentration dependent inhibition of the cellular accumulation of the radiopeptides. No significant differences in the uptake of the peptides between the control cells and those transfected with hOAT1 or hOCT2 were observed. ConclusionThe study showed that active transport mechanisms are decisive for the cellular accumulation in all tested 177Lu-labeled somatostatin, gastrin and bombesin analogues. Besides receptor-mediated endocytosis by megalin, FPE participates significantly in the uptake. The tested types of renal SLC transporters are not involved in this process.

CLN3 deficient cells display defects in the ARF1-Cdc42 pathway and actin-dependent events.

Juvenile Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) is a devastating neurodegenerative disease caused by mutations in CLN3, a protein of undefined function. Cell lines derived from patients or mice with CLN3 deficiency have impairments in actin-regulated processes such as endocytosis, autophagy, vesicular trafficking, and cell migration. Here we demonstrate the small GTPase Cdc42 is misregulated in the absence of CLN3, and thus may be a common link to multiple cellular defects. We discover that active Cdc42 (Cdc42-GTP) is elevated in endothelial cells from CLN3 deficient mouse brain, and correlates with enhanced PAK-1 phosphorylation, LIMK membrane recruitment, and altered actin-driven events. We also demonstrate dramatically reduced plasma membrane recruitment of the Cdc42 GTPase activating protein, ARHGAP21. In line with this, GTP-loaded ARF1, an effector of ARHGAP21 recruitment, is depressed. Together these data implicate misregulated ARF1-Cdc42 signaling as a central defect in JNCL cells, which in-turn impairs various cell functions. Furthermore our findings support concerted action of ARF1, ARHGAP21, and Cdc42 to regulate fluid phase endocytosis in mammalian cells. The ARF1-Cdc42 pathway presents a promising new avenue for JNCL therapeutic development.

Ultrasound Enhanced PEI-Mediated Gene Delivery Through Increasing the Intracellular Calcium Level and PKC-δ Protein Expression

Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. It has also been shown that combining ultrasound (US) with PEI could enhance and prolong in vitro and in vivo transgene expression. However, the role US in the enhancement of PEI uptake is poorly understood. This study investigates the impact of US on PEI-mediated gene transfection. Specific endocytosis pathway siRNA, including clathrin HC siRNA, caveolin-1 siRNA and protein kinase C-delta (PKC-δ) siRNA, are used to block the corresponding endocytosis pathways prior to the transfection of luciferase DNA/PEI polyplexes to cultured cells by 1-MHz pulsed US with ultrasound contrast agent SonoVue®. Transgene expression was found not to be enhanced by US treatment in the presence of the PKC-δ siRNA. We further demonstrated that PKC-δ protein could be enhanced at 6h after US exposure. Moreover, intracellular calcium levels were found to be significantly increased at 3h after US exposure, while transgene expressions were significantly reduced in the presence of calcium channel blockers both in vitro and in vivo. Our results suggest that US enhanced PEI-mediated gene transfection specifically by increasing PKC-δ related fluid phase endocytosis, which was induced by increasing the intracellular calcium levels.

Sequential breakdown of 3-phosphorylated phosphoinositides is essential for the completion of macropinocytosis

Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and solutes are internalized into cells. Macropinocytosis starts with the formation of membrane ruffles at the plasma membrane and ends with their closure. The transient and sequential emergence of phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 in the membrane ruffles is essential for macropinocytosis. By making use of information in the Caenorhabditis elegans mutants defective in fluid-phase endocytosis, we found that mammalian phosphoinositide phosphatase MTMR6 that dephosphorylates PI(3)P to PI, and its binding partner MTMR9, are required for macropinocytosis. INPP4B, which dephosphorylates PI(3,4)P2 to PI(3)P, was also found to be essential for macropinocytosis. These phosphatases operate after the formation of membrane ruffles to complete macropinocytosis. Finally, we showed that KCa3.1, a Ca(2+)-activated K(+) channel that is activated by PI(3)P, is required for macropinocytosis. We propose that the sequential breakdown of PI(3,4,5)P3 → PI(3,4)P2 → PI(3)P → PI controls macropinocytosis through specific effectors of the intermediate phosphoinositides.

Lysosomal enzymes may cross the blood–brain-barrier by pinocytosis: Implications for Enzyme Replacement Therapy

Here we hypothesized that the water-soluble lysosomal enzymes may cross the blood–brain-barrier and reach the brain using the mechanism of unspecific fluid-phase endocytosis. We also highlight studies that show that, at higher serum concentrations, a fraction of these proteins can reach the brain after intravenous injection, and we suggest some experiments to study this hypothesis. Finally we discuss the implications of this for treatments such as enzyme replacement of lysosomal storage disorders.

Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis.

Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB) on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10−8 cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

Detection of Small GTP Binding Proteins Showing GTPase and GTP/ATP Binding Activities in the Ovary of the American Cockroach, Periplaneta Americana, during Oogenesis

In the present study, the small GTP binding proteins involved in the regulatory mechanism of vitellogenin (Vg) endocytotic vesicles trafficking were detected, for the first time, in ovaries of the most basal hemimetabolus insect, Periplaneta americana during oogenesis. The ovarian GTPase activities were peaked during previtellogenic and early vitellogenic periods. Such activity coincides with vitellogenin receptors (VgRs) and clathrin early expression during these developmental periods, suggesting the importance of GTPases not only in the process of vesicle formation and fusion but also in the process of early fluid phase endocytosis. Two small peaks of activities were monitored during the late vitellogenic period (days 8 and 10), suggesting a possible role of GTPases in VgRs and clathrin recycling process. The [α32P]-GTP binding assay analysis in different tissues revealed the presence of small GTP binding proteins of molecular weights 25, 23 and 21 kDa in ovaries and head. However, a single binding signal band of 21 and 25 kDa was each detected in the fat bodies and muscles, respectively. No binding was detected in the midgut and Malpighian tubules. However, the 23 kDa protein detected was suggested as a probable cytosolic form of the 25 kDa protein. The competition assay results indicated that the small ovarian GTP binding proteins could also bind ATP, suggesting that like GTP, ATP is a regulatory nucleotide for the ovarian small proteins detected during oogenesis. The present study will pave the way for more understanding of the mechanisms that regulate Vg transport machinery in hemimetabolous insects.

Analysis of fluid-phase endocytosis in (intact) plant cells.

Endocytosis is a continuous process at the plasma membrane at least of all eukaryotic cells. Regardless of the molecular machinery, which drives the formation and uptake of endocytic vesicles, it is reasonable to assume that this process inevitably collects external fluid. Hence, at least for the majority of apoplastic solutes, the endocytosis of the fluid phase is likely to be an inevitable process. Due to its independence from the molecular machinery and low selectivity with respect to the cargo, it is thus perfectly suited to be used as a tracer to follow the activity of all endocytic events. Here we describe simple protocols based on fluorescence microscopy, which yield quantitative information about endocytic vesicle sizes-with sub-diffraction accuracy-as well as the size exclusion limits for these uptake routes.

Antibody Uptake into Neurons Occurs Primarily via Clathrin-dependent Fcγ Receptor Endocytosis and Is a Prerequisite for Acute Tau Protein Clearance

Tau immunotherapy is effective in transgenic mice, but the mechanisms of Tau clearance are not well known. To this end, Tau antibody uptake was analyzed in brain slice cultures and primary neurons. Internalization was rapid (<1 h), saturable, and substantial compared with control mouse IgG. Furthermore, temperature reduction to 4 °C, an excess of unlabeled mouse IgG, or an excess of Tau antibodies reduced uptake in slices by 63, 41, and 62%, respectively (p = 0.002, 0.04, and 0.005). Uptake strongly correlated with total and insoluble Tau levels (r(2) = 0.77 and 0.87 and p = 0.002 and 0.0002), suggesting that Tau aggregates influence antibody internalization and/or retention within neurons. Inhibiting phagocytosis did not reduce uptake in slices or neuronal cultures, indicating limited microglial involvement. In contrast, clathrin-specific inhibitors reduced uptake in neurons (≤ 78%, p < 0.0001) and slices (≤ 35%, p = 0.03), demonstrating receptor-mediated endocytosis as the primary uptake pathway. Fluid phase endocytosis accounted for the remainder of antibody uptake in primary neurons, based on co-staining with internalized dextran. The receptor-mediated uptake is to a large extent via low affinity FcγII/III receptors and can be blocked in slices (43%, p = 0.04) and neurons (53%, p = 0.008) with an antibody against these receptors. Importantly, antibody internalization appears to be necessary for Tau reduction in primary neurons. Overall, these findings clarify that Tau antibody uptake is primarily receptor-mediated, that these antibodies are mainly found in neurons with Tau aggregates, and that their intracellular interaction leads to clearance of Tau pathology, all of which have major implications for therapeutic development of this approach.

Inositol Polyphosphate Phosphatidylinositol 5-Phosphatase9 (At5PTase9) Controls Plant Salt Tolerance by Regulating Endocytosis

Phosphatidylinositol 5-phosphatases (5PTases) that hydrolyze the 5' position of the inositol ring are key components of membrane trafficking system. Recently, we reported that mutation in At5PTase7 gene reduced production of reactive oxygen species (ROS) and decreased expression of stress-responsive genes, resulting in increased salt sensitivity. Here, we describe an even more salt-sensitive 5ptase mutant, At5ptase9, which also hydrolyzes the 5' phosphate groups specifically from membrane-bound phosphatidylinositides. Interestingly, the mutants were more tolerant to osmotic stress. We analyzed the main cellular processes that may be affected by the mutation, such as production of ROS, influx of calcium, and induction of salt-response genes. The At5ptase9 mutants showed reduced ROS production and Ca(2+) influx, as well as decreased fluid-phase endocytosis. Inhibition of endocytosis by phenylarsine oxide or Tyrphostin A23 in wild-type plants blocked these responses. Induction of salt-responsive genes in wild-type plants was also suppressed by the endocytosis inhibitors. Thus, inhibition of endocytosis in wild-type plants mimicked the salt stress responses, observed in the At5ptase9 mutants. In summary, our results show a key non-redundant role of At5PTase7 and 9 isozymes, and underscore the localization of membrane-bound PtdIns in regulating plant salt tolerance by coordinating the endocytosis, ROS production, Ca(2+) influx, and induction of stress-responsive genes.