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Published as doi: 10.1096/fj.05-4621fje.
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(The FASEB Journal. 2006;20:2399-2401.)
© 2006 FASEB

Regulation of transferrin recycling kinetics by PtdIns[4,5]P2 availability

Sunyun Kim*, Hyunmyung Kim*, Belle Chang{dagger}, Namhui Ahn*, Suha Hwang*, Gilbert Di Paolo{dagger} and Sunghoe Chang*,1

* Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, South Korea; and

{dagger} Department of Pathology, College of Physicians and Surgeons, Columbia University Medical Center, New York, New York, USA

1Correspondence: Department of Life Science, Gwangju Institute of Science and Technology, 1 Oryong-dong Buk-gu, Gwangju, South Korea. E-mail: sunghoe{at}gist.ac.kr

ABSTRACT

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) is a phosphoinositide involved in a variety of cellular functions, including signal transduction, organelle trafficking, and actin dynamics. Although the role of PtdIns[4,5]P2 in endocytosis is well established, the precise trafficking steps relying on normal PtdIns[4,5]P2 balance in the endosomal pathway have not yet been elucidated. Here we show that decrease in intracellular PtdIns[4,5]P2 levels achieved by the overexpression of the 5-phosphatase domain of synaptojanin 1 or by siRNA knock-down of PIP5Ks expression lead to severe defects in the internalization of transferrin as well as in the recycling of internalized transferrin back to the cell surface in COS-7 cells. These defects suggest that PtdIns[4,5]P2 participates in multiple trafficking and/or sorting events during endocytosis. Coexpression of the PtdIns[4,5]P2 synthesizing enzyme, PIP5KI{gamma}, was able to rescue these endocytic defects. Furthermore, decreased levels of PtdIns[4,5]P2 caused delays in rapid and slow membrane recycling pathways as well as a severe backup of endocytosed membrane. Taken together, our results demonstrate that PtdIns[4,5]P2 availability regulates multiple steps in the endocytic cycle in non-neuronal cells.—Kim, S., Kim, H., Chang, B., Ahn, N., Hwang, S., Di Paolo, G., Chang, S. Regulation of transferrin recycling kinetics by PtdIns[4,5]P2 availability.


Key Words: endocytosis • membrane recycling

PHOSPHOINOSITIDES PLAY AN essential role in modulation of a variety of cellular functions, including cytoskeletal dynamics and membrane trafficking (1) . A number of lipid interacting modules, such as epsin amino-terminal homology (ENTH), Fab1, YOTB, Vac1 and EEA1 (FYVE), band 4.1, ezrin radixin and moesin (FERM), pleckstrin homology (PH), and the phox homology (PX) domains, have been identified, and these modular interactions recruit cytosolic proteins to specific compartments in cells to tune up various cellular functions (2) .

Recent studies showed that PtdIns[4,5]P2 is nonuniformly distributed at the plasma membrane and may be located in raft-like domains (3 , 4) . PtdIns[4,5]P2 at these sites coordinates membrane fission and fusion reactions with actin filament assembly to promote membrane movement (4) . PtdIns[4,5]P2 is known to be essential for maintaining Golgi organization and regulating the budding of protein cargo from this organelle through the formation of transport vesicles (4) . In addition, PtdIns[4,5]P2 may have multiple roles in budding and fusion events on the sorting and recycling endosomes, where a pool of PtdIns[4,5]P2 may reside (4 , 5) .

Although the involvement of PtdIns[4,5]P2 in exocytosis is still unclear, there is strong evidence that PtdIns[4,5]P2 can directly affect clathrin-mediated endocytic processes. The endocytic pathway is composed of both sorting and recycling endosomes. Sorting endosomes separate endocytosed material that is to be recycled from material destined for the lysosome. Sorting endosomes proceed to the plasma membrane either directly or via recycling endosomes. PtdIns[4,5]P2 binds several endocytic components, such as activating protein AP-2, AP-180, dynamin, epsin, and is known to play an essential role in recruiting these molecules to the sites of endocytosis (6 7 8 9) . The polyphosphoinositide phosphatase synaptojanin 1, which largely functions as a PtdIns[4,5]P2 5-phosphatase in vivo, was shown to be required for a late step of the endocytic process and more specifically, for the uncoating of clathrin-coated vesicles (10 11 12 13) . Overexpression of the 5-phosphatase domain of synaptojanin (5-PPase) or the PH domain of PLC{delta}, which binds and sequesters PtdIns[4,5]P2, causes defects in clathrin-mediated endocytosis (14 , 15) . A recent study of PIP5KI{gamma} knockout mice has demonstrated a critical role for PtdIns[4,5]P2 synthesis in the regulation of synaptic vesicle endocytosis (16) . The discovery of vesicle propulsion within the cell by actin comets also revealed an important new role for PtdIns[4,5]P2 in membrane trafficking as a regulator of vesicle movement through mediating the directed assembly of actin (17) . Actin comets are found to be attached to endosomes, pinosomes, and clathrin-coated vesicles (17 , 18) .

In summary, PtdIns[4,5]P2 plays a general role in membrane trafficking including early endocytosis, endosomal trafficking, vesicle budding from endosome, and subsequent fusion with plasma membrane. However, the precise steps involving PtdIns[4,5]P2 in the endosomal pathway have not been thoroughly characterized. In this study, we provide evidence that PtdIns[4,5]P2 availability regulates multiple steps in the endocytic recycling pathway.

MATERIALS AND METHODS

Cell culture and Transfection
COS-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% NCS [~120,000 cells in a 60 mm petri-dish for imaging, ~150,000 cells in a 60 mm petri-dish for thin layer chromatography (TLC)] and maintained at 37°C in a 95% air, 5% CO2 humidified incubator. Transfections were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with 5-PPase, PLC{delta}-PH or PBP. For rescue experiments, 5-PPase and PIP5KI{gamma} were cotransfected into cells. All constructs were GFP tagged with the exception of PIP5KI{gamma}, which was hemagglutinin (HA) tagged. E-18 primary rat hippocampal neurons were prepared and transfected using a calcium phosphate method as described previously (19) . Media and supplements were from Invitrogen and all other chemicals were from Sigma (St. Louis, MO).

In vitro enzyme assay and TLC
Cells transfected with 5-PPase alone or cotransfected with PIP5KI{gamma} were lysed in extracting buffer (1% TritonX-100, 100 mM KCl, 1 mM MgCl2, and 30 mM HEPES, pH 7.4). After 20 min incubation on ice, lysates were centrifuged and supernatants were collected; 20 µg of cell extracts in reaction buffer (50 mM ammonium carbonate, 2 mM dithiothreitol, and 200 µM ATP, pH 8.0) were incubated with 1 µg of nitro benzoxadiazole (NBD)6-PtdIns[4,5]P2 (Echelon Bioscience, Salt Lake City, UT) for 20 min at 37°C, and the reaction was terminated by adding acetone. Reaction products were dried down in a Speed-Vac, resuspended in methanol/2-propanol/glacial acetic acid (5/5/2), and spotted onto TLC plates (K6 silica gel 60 Å, Whatman, Clifton, NJ). Phosphoinositides were separated in chloroform/methanol/water/ammonium hydroxide (60/47/11.3/2). PtdIns[4,5]P2 and PtdIns[4]P were quantified in NucleoVision Imaging Workstation with GelExpert software (Nucleotech, Westport, CT). Fluorescence intensities of bands were normalized to the sum of fluorescence intensities of each group.

Transferrin uptake assay
Twenty-four hours after transfection, COS-7 cells were starved for 6–8 h in serum-free DMEM with 0.1% BSA and incubated in serum-free DMEM/HEPES containing 20 µg/ml Texas red transferrin (Molecular Probe, Eugene, OR) for 10 min at 37°C. Cells were washed in acid stripping solution (150 mM NaCl, 2 mM CaCl2, and 25 mM CH3COONa, pH 4.5) and fixed in 4% paraformaldehyde. To study transferrin recycling after continuous uptake, cells were incubated at 37°C in serum-free DMEM/HEPES containing 20 µg/ml Texas red transferrin for 60 min. After acid stripping, DMEM/HEPES containing 100 µg/ml unlabeled transferrin (Sigma) was added and the cells were incubated at 37°C for various lengths of time. To study a single round of Texas red transferrin endocytosis and recycling, cells were incubated on ice in serum-free DMEM/HEPES containing 20 µg/ml of Texas red transferrin for 1 h, washed with ice-cold DMEM/HEPES, and then incubated at 37°C for various lengths of time. At each time point, cells were washed with ice-cold PBS, acid-stripped, and fixed immediately. Images were taken on a Olympus IX-71 microscope (Olympus Corp., Tokyo, Japan) using a x40 N.A. 1.0 objective and CoolSNAP-Hq CCD camera (Roper Scientific, Tucson, AZ). Texas red intensities were averaged over individual cells and analyzed using MetaMorph (Universal Imaging Corp., West Chester, PA).

Immunostaining of transferrin receptor
Twenty-four hours after transfection, COS-7 and HeLa cells were fixed in 4% paraformaldehyde and incubated overnight at 4°C in transferrin-receptor antibody (Ab) diluted 1:1000 in PBS with 3% BSA. After incubation in primary Ab, cells were incubated for 45 min at 37°C in secondary Ab (anti-mouse Texas red) diluted 1:1000 in PBS with 3% BSA. Cells were not permeabilized to measure the amount of transferrin-receptors on surface area.

Dextran and epidermal growth factor (EGF) uptake assay
Twenty-four hours after transfection, COS-7 and HeLa cells were starved for 4 h in serum-free DMEM with 0.1% BSA; 1 mg/ml of Texas red dextran in PBS with 10% FBS was applied to cells on ice and incubated for indicated times at 37°C. After incubation, cells were fixed in 4% paraformaldehyde and observed.

For EGF uptake assay, 500 ng/ml of Texas red EGF in binding buffer (20 mM HEPES-NaOH, 130 mM NaCl, and 0.1% BSA, pH 7.5) was applied to cells and incubated for 1 h at 4°C after starvation to saturate EGF receptors on the cell surface. Next, cells were incubated in serum-free DMEM with 0.1% BSA for 15 min at 37°C. Cells were then fixed in 4% paraformaldehyde.

Actin and tubulin immunostaining
COS-7 and HeLa cells were fixed in 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 in PBS. Cells were then stained for 45 min at 37°C in Texas red-X phalloidin diluted in PBS with 3% BSA to observe actin structure.

For tubulin staining, cells were preincubated in microtubule stabilizing buffer (80 mM PIPES, 1 mM MgCl2, and 4 mM EGTA, pH 6.8) with 10 µM of taxol and fixed in 0.5% glutaldehyde. Subsequently, cells were permeabilized with 0.1% Triton X-100 in PBS and incubated overnight at 4°C in ß-tubulin Ab diluted 1:1000 in PBS with 3% BSA. After incubation in primary Ab, cells were incubated for 45 min at 37°C in secondary Ab (anti-mouse Texas red) diluted 1:1000 in PBS with 3% BSA.

Measurement of levels in PIP5KI-{alpha}, -ß, -{gamma} siRNA-treated cells
HeLa cells were transfected with either three PtdIns[4,5]P2/PIP5KI-{alpha} siRNAs or vector control. 48 h after transfection, the cells were subjected to two rounds of zeocin (Invitrogen) selection in DMEM/10% FCS/1 mg/ml zeocin to enrich for transfected cells. The cells were harvested 48 h after the final media change. To determine the anionic phospholipid content of these transfected cells, a variation of a published protocol was used (20) . Briefly, HeLa cells in 10-cm2 dishes were harvested in 320 mM sucrose, 5 mM HEPES, pH 7.4 buffer, spun down, and lysed with ice-cold lipid extraction solution CHCl3:MeOH:10N HCl (20:40:1) supplemented with 2 mM AlCl3. Lipids were then washed with ice-cold MeOH: 2mM oxalic acid (1:0.9), dried under N2 stream, and then deacylated by incubation with 0.5 ml methylamine reagent (MeOH: 40% methylamine in water: n-butanol: water: 47:36:9:8) at 50°C for 45 min. The aqueous phase was dried, resuspended in 0.5 ml of n-butanol:petroleum ether:ethyl formate (20:40:1), and extracted twice with an equal volume of water. Aqueous extracts were dried, resuspended in water, and subjected to anion-exchange HPLC on an Ionpac AS11-HC column (Dionex, Sunnyvale, CA). Negatively charged glycerol head groups were eluted with a 1.5–86 mM KOH gradient and detected online by suppressed conductivity (20) in a Dionex Ion Chromatography system equipped with an ASRS-ultra II self-regenerating suppressor. Individual peaks were identified and peak areas were calculated using the Chromeleon software (Dionex). With the use of deacylated anionic phospholipids as standards, lipid masses were calculated and expressed as molar fractions of total anionic phospholipids present in the sample. Results were expressed as the mean ratio of PtdIns[4,5]P2 to PtdIns[4]P levels (in mol %) for duplicate samples.

Kinetic Measurement of FM 5–95 trafficking
Recycling kinetic measurement of FM 5–95 trafficking was performed as described previously with a minor modification (21) . Briefly, control and 5-PPase transfected cells were labeled with 15 µM of FM 5–95 (Molecular Probes, Eugene, OR) for 1 min, washed with ice-cold Tyrode’s solution (136 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4), with 2 g/l Glc, and kept on ice for 15 min. Immediately before image acquisition, cells were mounted in a temperature controlled observation chamber (AC-S-10, LCI, Seoul, Korea) on the stage of a Olympus IX-71 microscope and perfused with 30°C Tyrode using an inline heating system(IHS-101, LCI). Images were acquired with a x40 N.A. 1.4 lens using a CoolSNAP-Hq CCD camera driven by MetaMorph with GFP/FM 5–95 optimized filter sets (Omega Optical, Brattleboro, VT). Analysis was carried out using SigmaPlot (Systat Software, Point Richmond, CA).

RNA interference
The siRNA sequences targeting PIP5KI{alpha}, PIP5KIß, and PIP5KI{gamma} were from position 1923–1943 (GenBank/EMBL/DDBJ accession no. U78575), 1114–1135 (GenBank/EMBL/DDBJ accession no. NM_003558), and 619–639 (GenBank/EMBL/DDBJ accession no. XM_047620), respectively. The annealed cDNA fragment with siRNA was cloned into the Acc65I-HindIII sites of the psiRNA-hH1GFPzeo G2 vector (InvivoGen, San Diego, CA). HeLa cells were transfected with each PIP5KI siRNA using Lipofectamine 2000 according to the manufacturer’s instructions and analyzed by immunoblot and endocytosis assay.

FM endocytosis/exocytosis assay
For FM endocytosis/exocytosis assays in cultured hippocampal neurons, coverslips were mounted in a stimulation chamber (ES-S-10, LCI) on the stage of a Olympus IX-71 fluorescence microscope. Cells were continuously perfused with a Tyrode’s solution containing 1% BSA at a rate of 1 ml/min at room temperature; 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 µM D-aminophosphonovalerate (APV) were added to the Tyrode’s solution to reduce spontaneous activity and prevent recurrent excitation during stimulation. Imaging was performed with a 40X N.A. 1.0 objective using a CoolSNAP-ES CCD camera driven by MetaMorph software with FM 5–95 optimized filter set. Analysis was carried out using MetaMorph and SigmaPlot. For the endocytosis assay, FM 5–95 was used at a concentration of 15 µM. FM 5–95 loading was performed by superfusing the dye into the chamber and electrically stimulating the neuron for 30 s at 10 Hz by passing current pulses between platinum electrodes of the chamber. After additional incubation for 30 s in the presence of FM 5–95, the dye was washed for 10 min in dye-free Tyrode’s solution, images were taken, and neurons were then stimulated for 2 min at 10 Hz to unload FM 5–95, and then images were taken again. After 10 min recovery, the second round FM loading was performed either in the same way as the first round (no delay) or with 20 s delay after the electrical stimulation began. In the 20 s before the onset of FM 5–95 application, some vesicles can undergo endocytosis, escaping the labeling, and thus remain unlabeled. Images were taken before and after unloading as depicted in Results. The second round fluorescence intensities ({Delta}F2=C-D) were normalized to the first round fluorescence intensities ({Delta}F1=A-B), averaged, and expressed as a ratio ({Delta}F2/{Delta}F1) over square regions centered on presynaptic boutons.

For exocytosis assays, FM 5–95 was loaded by superfusion and electrical stimulation of 10 Hz for 60 s. After additional incubation for 30 s in the presence of FM 5–95, the dye was washed away for 10 min, and an electrical stimulation of 10 Hz for 2 min was applied to release vesicular dye, while images were taken every 10 s as depicted in Results. Quantitative measurements of fluorescence intensities were obtained over square regions centered on presynaptic boutons and normalized to the initial resting state of each presynaptic bouton. The decay of fluorescence was fitted with a single exponential using SigmaPlot. The time constant ({tau}) of each presynaptic bouton was calculated and averaged.

RESULTS

Overexpression of 5-PP decreased PtdIns[4,5]P2 levels
As a prelude to our functional studies, we tested whether the overexpression of the 5-PPase in COS-7 cells could significantly affect PtdIns[4,5]P2 metabolism. After the transfection of COS-7 cells with 5-PPase construct, cytosolic extracts prepared from transfected cells were incubated with the lipid substrate NBD6-PtdIns[4,5]P2 in the presence of ATP and reaction products were analyzed by TLC. The TLC results show that there is a reciprocal relation between PtdIns[4,5]P2 and PtdIns[4]P levels. In the extract from cells overexpressing 5-PPase, PtdIns[4,5]P2 substrate levels were decreased by converting it to PtdIns[4]P (control vs. 5-PPase; PtdIns[4,5]P2: 49.7 vs. 24.3%; PtdIns[4]P: 50.3 vs. 75.7%, Fig. 1 ). We tested whether the coexpression of PtdIns[4]P 5-kinase type I{gamma} (PIP5KI{gamma}) with 5-PPase could restore normal PtdIns[4,5]P2 levels. Figure 1 shows that PtdIns[4,5]P2 substrate levels were restored on coexpression of PIP5KI{gamma} and 5-PPase in COS-7 cells (control vs. coexpression; PtdIns[4,5]P2: 49.7 vs. 41.7%; PtdIns[4]P: 50.3 vs. 58.3%). The same trend was observed when phosphoinositide levels were measured using anion-exchange HPLC with suppressed conductivity detection.


Figure 1
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  		Figure 1. Overexpression of 5-PP decreases PtdIns[4,5]P2 levels. A) Picture of TLC showing lipid products generated by incubation of cell cytosolic fractions with NBD6-PtdIns[4,5]P2. Fluorescence intensities associated with PtdIns[4]P and PtdIns[4,5]P2 was quantified and are shown in B. Black bar: PtdIns[4]P; gray bar: PtdIns[4,5]P2. 5PP: 5-phosphatase of synaptojanin, PIPK: PIP5KI{gamma}. Data are mean ± SD; n = 5.

Transferrin endocytosis and recycling are impaired by the decrease of PtdIns[4,5]P2 level
Increasing evidence suggests that PtdIns[4,5]P2 is directly involved in the regulation of trafficking steps to and from the plasma membrane, although the precise steps relying on proper PtdIns[4,5]P2 balance remain to be identified.

To test the role of PtdIns[4,5]P2 in endocytosis, we first performed a transferrin uptake assay. COS-7 cells were transfected with the 5-PPase, PLC{delta} 1-PH or PBP and then allowed to take up Texas red conjugated transferrin for 10 min at 37°C. 5-PPase dephosphorylates PtdIns[4,5]P2 into PtdIns[4]P, while PLC{delta}-PH and PBP bind PtdIns[4,5]P2 with high affinity, thereby resulting in PtdIns[4,5]P2 sequestration (14 , 22 23 24 25) .

In control, nontransfected cells, Texas red transferrin was internalized and accumulated in the perinuclear endosomal compartment and in punctate structures dispersed throughout the cytoplasm, whereas in cells overexpressing 5-PPase, PLC{delta}-PH, or PBP, no such accumulation was observed (average Texas red transferrin fluorescence in a.f.u: 5-PPase, 54.0±12.4; PLC{delta}-PH, 78.3±8.1; PBP, 88.4±8.7; control, 173.4±7.0, Fig. 2 ). Cells transfected with 5-PPase showed the most significant inhibition in transferrin uptake (~70% reduction in the uptake of transferrin) among the three experimental groups probably because the elimination of PtdIns[4,5]P2 achieved by the enzymatic activity is more effective than that obtained by sequestration of this lipid through PtdIns[4,5]P2-binding modules.


Figure 2
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  		Figure 2. Inhibition of transferrin uptake as a result of decreased PtdIns[4,5]P2 levels. A, right panel) GFP image. A, left panel) Texas red transferring. A, middle) Merged images. In cells expressing 5PP, PBP, or PH, transferrin uptake was dramatically reduced. Coexpression of 5PP with PIPK rescued defects in transferrin uptake. Averaged fluorescence intensity of Texas red transferrin was quantified and is shown in B. C) Surface binding capacity of transferrin was quantified in COS-7 cell with averaged fluorescence intensity of transferrin receptor immunostaining. Cells were fixed and processed as described in Materials and Methods. All images were collected with 1 s exposures. PBP: PtdIns[4,5]P2 binding peptide of gelsolin, PH: PLC{delta}-PH domain, PIPK: PIP5KI{gamma}. *Statistically significant at P < 0.01; P = 0.02 for 5-PP+PIPK, Student’s t test. Bar = 20 µm.

Defects in receptor-mediated endocytosis resulting from decreased PtdIns[4,5]P2 levels were also confirmed using the independent endocytosis marker EGF (Supplemental Fig. 1A ). In cells expressing 5-PPase, the uptake of EGF was reduced relative to control (average Texas red EGF fluorescence in a.f.u: 5-PPase, 75.41±3.89; control, 50.1±2.07).

In contrast, the uptake of an unrelated fluid phase endocytic marker, Texas-red dextran, was not affected by 5-PPase overexpression, suggesting that normal PtdIns[4,5]P2 balance is not required for all forms of endocytosis (Supplemental Fig. 1C).

Our biochemical experiments show that the level of PtdIns[4,5]P2 was recovered to the degree that is comparable to that of the control cells when PIP5KI{gamma} was coexpressed with 5-PPase (Fig. 1) . If the defect in transferrin endocytosis observed in 5-PPase overexpressed cells is due to a decrease in the levels of PtdIns[4,5]P2 as we hypothesize, coexpression of PIP5KI{gamma} with 5-PPase should rescue the endocytic defects. This was indeed the case. Figure 2A and B , shows that transferrin uptake and endocytosis were reduced with 5-PPase overexpression (54.0 a.f.u. ± 12.4) and were recovered to a level comparable to that of the control group (average Texas red transferrin fluorescence in a.f.u: 173.4±7.0 for control vs. 139.3±10.0 for cotransfection). Theoretically, the decrease in transferrin internalization observed in 5-PPase expressed cells may originate from a mere decrease in the surface binding capacity for transferrin. To rule out this possibility, we performed an immunofluorescence staining of transferrin receptors on nonpermeabilized cells, so that only the pool of receptors present at the cell surface could be detected. Figure 2C shows that there is no significant difference in the fluorescence associated with transferrin receptor at the cell surface in 5-PPase overexpressed cells compared to controls, suggesting a similar surface transferrin binding capacity.

Transferrin recycles through a series of endosomal compartments. After binding to its receptor, the receptor-bound transferrin is subjected to endocytosis. It is rapidly delivered to the sorting endosome and subsequently trafficked to a recycling compartment, from which it returns to the plasma membrane (26) . Therefore, we performed a single-round of transferrin uptake and recycling assay to test whether decrease in the level of PtdIns[4,5]P2 by 5-PPase expression could affect endocytosis as well as other aspects of transferrin recycling. In control cells, transferrin was endocytosed rapidly within 5 min, and the internalized transferrin was efficiently recycled (half-time ~10 min) back to the plasma membrane (Fig. 3 A). In contrast, 5-PPase expressed cells showed significant endocytosis and recycling defects. The initial rate of transferrin uptake decreased ~2-fold relative to the control (Fig. 3A , inset), and a significant fraction of the internalized transferrin still remained in the cells >60 min after uptake, in contrast to control cells, which had <20% of the initial fluorescence remaining (Fig. 3A ).


Figure 3
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  		Figure 3. Transferrin endocytosis and recycling are impaired by decreased PtdIns[4,5]P2 levels. A) Single-round kinetics of transferrin uptake and recycling were determined by first incubating cells with Texas red transferrin for 30 min on ice. After washout, cells were incubated in chase medium at 37°C for various times. At each time point, cells were fixed and processed as described in Materials and Methods. Inset expands data collected within initial 5 min. Results were normalized to total bound value and expressed as % of total bound. Data are mean ± SD; n = 5. B) Recycling of transferrin from endosomes was measured by first loading cells with Texas red transferrin at 37°C for 60 min to saturate the entire endocytic/recycling pathway. Surface bound Texas red transferrin was removed by acid-stripping and cells were reincubated at 37°C in chase medium for various times. In each experiment, results were normalized so that fluorescence value for Texas red transferrin at 0 min was 1.00. Inset expands data collected within initial 10 min. Data are mean ± SD; n = 5.

Although our results suggest that the decrease of PtdIns[4,5]P2 causes defects in transferrin recycling as well as endocytosis, the delay in transferrin recycling back to the plasma membrane in 5-PPase expressed cells could simply be due to the initial delay in transferrin internalization. To rule out this possibility, cells were incubated with Texas red transferrin for 60 min at 37°C to saturate the entire recycling pathway before measuring recycling kinetics. Figure 3B shows that the internalized transferrin was efficiently recycled back to the cell surface in control cells whereas 5-PPase expressed cells showed significant recycling defects in agreement with data obtained from our single-round transferrin recycling assays (half-time for control: 6.32 min, single exponential regression from 0 min; half-time for 5-PPase: 11.86 min, single exponential regression from 10 min). Even after 60 min incubation, cells expressing 5-PPase still retained ~40% of internalized Texas red transferrin whereas <25% of internalized Texas red transferrin remained in control cells (Fig. 3B ). When we compared the first 10 min of recycling kinetics (Fig. 3B inset), control cells released more than half of the internalized Texas red transferrin (44.9% of initial fluorescence remained) whereas 5-PPase expressed cells retained most of internalized Texas red transferrin (91.8% of initial fluorescence remained). After 10 min of lag phase, 5-PPase expressed cells started to release transferrin although at a slower rate than control.

We further confirmed our results using RNA interference of PIP5KIs. A recent study showed that RNA interference (RNAi) against each of the three known PIP5KI isoforms successfully decreases the levels of plasma membrane as well as intracellular PtdIns[4,5]P2 (27) . This study, however, showed that gene expression of each PIP5KI isoform is sensitive to the expression of the other isoforms in HeLa cells. For example, RNAi targeting PIP5KIß slightly reduced PIP5KI{alpha} and induced a threefold increase in expression of PIP5KI{gamma}. Furthermore, siRNA targeting PIP5KI{alpha} caused a 2-fold increase in mRNA for both PIP5KIß and {gamma} (28) . Thus, to decrease PIP5KIs expression levels without the involvement of interdependency, we triply transfected HeLa cells with all three siRNA constructs targeting PIP5KI{alpha}, PIP5KIß, and PIP5KI{gamma} together.

Suppression of three PIPKI isoforms expression by siRNA was confirmed by immunoblotting, whereas expression of tubulin was shown to be unaffected (Fig. 4 A). In agreement with data obtained from our 5-PPase transfected cells, triply transfected cells showed significant recycling defects as well as endocytic defects as compared to the control (Fig. 4D ). Although the surface binding capacity of transferrin did not show any significant difference between control and triply transfected cells (Fig. 4B ), triply transfected cells were unable to take up transferrin as efficiently as control even with 60 min incubation, and the internalized transferrin was not efficiently recycled back to the cell surface (half-time for control: 6.32 min, single exponential regression from 0 min; half-time for siRNAs: 11.66 min, single exponential regression from 10 min). Interestingly, the time course of transferrin recycling obtained with siRNA experiments was almost identical to those obtained with 5-PPase transfections (half-time for 5-PPase: 11.86 min; half-time for siRNAs: 11.66 min). Furthermore, as in the case of 5-PPase expressed cells, there was a 10 min lag phase before triply transfected cells started to release transferrin. Consistent with our results on transferrin uptake, the internalization of EGF was also impaired in triply transfected cells (average Texas red EGF fluorescence in a.f.u: siRNA, 10.66±0.75; control, 20.02±0.94, Supplemental Fig. 1B).


Figure 4
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  		Figure 4. Reduced expression of PIP5KIs by RNA interference reduces rate of transferrin recycling. HeLa cells were triply transfected with siRNAs targeting the {alpha}, ß, and {gamma} isoform of PIP5KI together. Western blots with antibodies specific for each isoform and tubulin were performed 48 h after transfection. A) An example of a Western blot specific for each isoform of PIP5KI is shown. B) Surface binding capacity of transferrin was quantified in HeLa cell with averaged fluorescence intensity of transferrin receptor immunostaining. C) Ratio of PtdIns[4,5]P2 to PtdIns[4]P in control and in triply transfected cells measured by anion-exchange HPLC with suppressed conductivity detection. D) Recycling of transferrin from endosomes was measured by first loading cells with Texas red transferrin at 37°C for 60 min to saturate the entire endocytic/recycling pathway. Surface bound Texas red transferrin was removed by acid-stripping and cells were reincubated at 37°C in chase medium for various times. Averaged fluorescence intensity of Texas red transferrin was quantified and expressed as a.f.u. In inset, results were normalized so that value for transferrin at 0 min was 1.00. Data are mean ± SD; n = 7.

As seen with a decrease in the level of PIP5KI isoforms seen by Western blotting, the triply siRNA transfected cells showed an ~30% decrease in the mass of PtdIns[4,5]P2 and a 25% increase in that of PIP5KIs’ substrate PtdIns[4]P, as measured by anion-exchange HPLC with suppressed conductivity detection. Consequently, there was an ~45% decrease in the PtdIns[4,5]P2-to-PtdIns[4]P ratio (Fig. 4C ). Since the percentage of transfected cells was estimated to be ~50%, both with control and siRNA vectors, this result represents an underestimation of the actual decrease occurring in transfected cells.

Cell viability may be compromised by triple siRNA transfections, thereby causing a general impairment of endocytosis. To rule out this possibility, we first performed an endocytosis assay using Texas red-conjugated dextran and found no difference in the uptake of this tracer between control and siRNAs transfected cells (Supplemental Fig. 1D ). Additionally, we stained actin and microtubules using phalloidin and a specific tubulin Ab to test whether triple siRNA transfection caused a global perturbation of cellular structures. We did not observe any significant structural abnormalities in triply transfected cells (Supplemental Fig. 2A, B ).

Taken together, these data suggested that the decrease in PtdIns[4,5]P2 availability caused defects not only in internalization of transferrin but also in recycling of transferrin through endosomal compartments back to the cell surface.

Membrane recycling is impaired by the decrease of PtdIns[4,5]P2 level
Lipids and other membrane constituents also recycle between the plasma membrane and intracellular endocytic compartments. The existence of an alternate, fast recycling pathway, in addition to the slow recycling pathway previously characterized, has been suggested (26 , 29) . Hao and Maxfield (21) have further characterized the rapid recycling pathway using fluorescent lipophilic dyes, the FM dyes, and showed that the fast recycling pathway involves vesicular transport from the sorting endosome to the plasma membrane, whereas the slow pathway involves transport from the recycling endosome. Consequently, our next goal was to test whether reduction of PtdIns[4,5]P2 availability would affect any of these two membrane recycling pathways toward the plasma membrane. The FM dyes are virtually nonfluorescent in aqueous medium and become intensely fluorescent on binding to the membrane. Taking advantage of this property, we used a live cell imaging to study the efflux kinetics of the FM dyes. The efflux kinetics of FM dyes represents the recycling kinetics of membrane constituents back to the plasma membrane. We have used FM 5–95 that is more hydrophobic compared to FM 1–43, since the efflux kinetics of FM 5–95 show a biphasic kinetic profile similar to that using FM 1–43 (21) . Cells were first labeled with FM 5–95 for 1 min, and the efflux kinetics of FM 5–95 was imaged for 60 min. In control cells, data fit well with a double exponential decay, giving a half-time of ~2.5 min for the fast component and ~23 min for the slow component (R2=0.997, (Fig. 5 ). In cells expressing 5-PPase, however, half-times for the fast and slow component were slightly but significantly longer than those of control cells (3.7 min for the fast component, 27.4 min for the slow component; R2=0.991, Fig. 5 ). More importantly, 5-PPase overexpressing cells retained a higher fraction of residual FM 5–95 fluorescence after 60 min incubation (24.2% for control, 42.1% for 5-PPase). These data suggest both the rapid pathway from the sorting endosome and the slow pathway from the recycling endosome are delayed and a significant fraction of FM 5–95 was "trapped" in the endosomal recycling pathway in 5-PPase expressed cells. The ratio of fast component to slow component was not significantly different between control and 5-PPase transfected cells (34%: 66% for control; 36%: 64% for 5-PPase). Taken together, our results suggest that PtdIns[4,5]P2 plays a critical role after membrane internalization in one or multiple steps of the membrane recycling pathway.


Figure 5
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  		Figure 5. Membrane recycling is impaired by decrease in PtdIns[4,5]P2. Cells were labeled with FM 5–95 for 1 min at 37°C, washed with ice-cold Tyrode, warmed up to 37°C, and imaged for 60 min. Efflux kinetic curves of FM 5–95 after a 1 min pulse were obtained by quantification of fluorescent images. Fluorescent values were normalized to that of the first value at beginning of chase. Data points fit well to a double exponential decay (R2>0.99), giving a half-time of ~2–3 min for fast component and ~20–25 min for slow component. Note that after 60 min incubation, substantial amount of fluorescence was still retained in 5-PPase expressed cells. Data are mean ± SD; n = 4.

Synaptic vesicle endocytosis slows down by the decrease of PtdIns[4,5]P2 level
To validate our results obtained with the transferrin uptake experiments, we tested whether the tools used to manipulate PtdIns[4,5]P2 levels in COS-7 cells could also affect synaptic vesicle recycling in nerve terminals from primary hippocampal cultures, in agreement with previous genetic studies (16) . Dissociated hippocampal neurons were isolated, and transfected with the construct expressing 5-PPase, PLC{delta}-PH, or PBP. To measure the rate of synaptic vesicle endocytosis, we used FM 5–95 that is selectively taken up into synaptic vesicles during electrical stimulation. The strategy used for this study is depicted in Fig. 6 A. Two rounds of staining and destaining were performed on the same microscopic field of each coverslip. The electrical stimulations to load or to unload the dye were given in identical conditions between the first and the second round. By the application of FM dye and electrical stimulation at the same time across two rounds of staining and destaining, it was confirmed that 10 min of recovery time was enough for cells to fully recovery to the initial physiological condition, and the recycling vesicle pool of each bouton was identical across two rounds of loading and unloading. Then, at the second round, the FM dye was added with a 20 s delay after the beginning of an electrical stimulation. In this procedure, recycled vesicles before the application of FM dye will escape from staining. Therefore, the more slowly vesicles recycle, the more vesicles will be stained with FM dye. Only the boutons loaded with FM dye that exactly overlaps the GFP signal of axon were selected as an experimental group, and adjacent boutons were analyzed as its counter group.


Figure 6
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  		Figure 6. Synaptic vesicle endocytosis slows down by reduction of PtdIns[4,5]P2 availability. A) Strategy used for measuring rate of synaptic vesicle endocytosis. Neurons were transfected with each construct and two consecutive load-unload cycles with FM 5–95 were performed (the first cycle without delay and the second cycle with 20 s delay). See text for detailed description of experiments. B) FM 5–95 loading intensities were expressed as ratio of intensity of first load and averaged across synaptic population. Cells transfected with each construct, 5PP, PLC{delta}-PH or PBP, showed endocytic defects as compared to the control group. Recovery of endocytosis defect was observed in cells that were cotransfected with 5PP and PIPK. Filled circle are data from control group in each experiment while open triangle represents data from transfected boutons with each construct. PBP: PtdIns[4,5]P2 binding peptide of gelsolin, PLC{delta}-PH domain, PIPK: PIP5kinase type I{gamma}. Data are mean ± SD; *significantly different from the control group. P values and number of boutons analyzed were indicated in Table 1 .

Figure 6B shows that cells transfected with 5-PPase, PLC{delta}-PH, or PBP, all showed defects in synaptic vesicle endocytosis. With 20 s delay in the second loading, the cells expressing each construct contain an invariably higher fraction of FM 5–95 than the control cells, indicating the slow down of synaptic vesicle endocytosis. The ratio of FM load ({Delta}Fsecond/{Delta}Ffirst) was 0.80 ± 0.06 for 5-PPase, 0.79 ± 0.03 for PLC{delta}-PH, and 0.77 ± 0.015 for PBP, while 0.63 ± 0.03, 0.63 ± 0.02, and 0.66 ± 0.01 for each control group, respectively (Fig. 6B and Table 1 ).


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  		Table 1. Summary of FM load ratio in the endocytosis assay

As in the case of COS-7 cells, the coexpression with PIP5KI{gamma} successfully overcame the endocytosis defect caused by the expression of 5-PPase (Fig. 6B ). The ratio of FM load in the cotransfected group was not significantly different from its control group (0.70±0.04 and 0.64±0.05, respectively, P=0.37, Fig. 6B and Table 1 ). Thus, our results clearly show that a decrease in PtdIns[4,5]P2 levels slows down the rate of endocytosis, consistent with our results reported for the transferrin uptake as well as those obtained in cultured PIP5KI{gamma}–/– cortical neurons using the genetically-encoded fluorescent probe, synaptopHluorin (16) . In agreement with the genetic study on PIP5KI{gamma}, we did not observed any defect of exocytosis using styryl dye uptake and release assays (Supplemental Fig. 3; see also ref 16 ).

DISCUSSION

In the current study, we have unmasked for the first time the role of PtdIns[4,5]P2 in multiple trafficking and/or sorting events of transferrin in non-neuronal cells. Previous studies including PIP5KI overexpression and RNA interference studies have shown a role for PtdIns[4,5]P2 in the process of transferrin uptake, although the precise trafficking steps relying on normal PtdIns[4,5]P2 balance were not addressed (28) . Our results clearly demonstrate that the initial step of endocytosis, which largely relies on clathrin-mediated endocytosis, is not the only process affected by changes in PtdIns[4,5]P2 metabolism. Indeed, our dye uptake and release assays have shown that the recycling of transferrin back to the plasma membrane is also severely affected in cells containing lower levels of PtdIns[4,5]P2. Strikingly, both the slow and the fast membrane recycling pathways were altered, indicating a more general impairment in vesicular trafficking. The fast pathway involves transport from the sorting endosome to the plasma membrane while the slow pathway involves transport from the recycling endosome. Therefore, the fact that the rate of both the fast and the slow components were slightly but significantly slowed down by 5-PPase expression and that the ratio of fast component to slow component was not significantly different between control and 5-PPase transfected cells suggests that both pathways were affected roughly to the same extent by the decrease in PtdIns[4,5]P2 levels. Thus the endocytosed transferrin/membranes may be trapped both in the sorting endosome and in the recycling endosome. Although FM dye could be taken up by nonreceptor mediated endocytosis, since a high fraction of internalized C6-NBD-SM or FM dyes is recycled to the cell surface through an itinerary that is morphologically and kinetically indistinguishable from recycling transferrin receptors, we could assume that a considerably high fraction of FM dye enters the cells through receptor-mediated endocytosis.

The enrichment of PtdIns[4,5]P2 at the plasma membrane suggests that the trafficking steps suffering the most from a reduction in PtdIns[4,5]P2 levels might be the endocytic reaction per se, as shown in various studies (11 , 15 , 16 , 28 , 30) . Alternatively, budding and fusion events on the sorting and recycling endosomes may rely on normal PtdIns[4,5]P2 balance, where a small, but functionally important, pool of PtdIns[4,5]P2 may reside. Indeed a recent study (16) of PIP5KI{gamma} knockout study has revealed the defects in the multiple steps of synaptic vesicle trafficking, including a severe back-up of endocytic traffic both in the plasma membrane and in endosome-like structures.

PtdIns[4,5]P2 is thought to be essential for the exit of protein cargo from the Golgi by the formation of transport vesicle which requires PtdIns[4,5]P2 (31) . In addition to vesicle budding, PtdIns[4,5]P2 may participate in a step linked to fusion (4 , 5) . Since the recycling of membrane material involves several budding and fusion steps as well as transport steps, the identification of the precise trafficking steps impaired in cells containing lower levels of PtdIns[4,5]P2 will require additional work. Finally, based on the known effects of PtdIns[4,5]P2 on the actin cytoskeleton (32 , 33) , some of the effects observed in PtdIns[4,5]P2-deficient cells may be due to defects in the actin cytoskeleton, since the uptake and recycling of transferrin has been shown to rely on normal actin dynamics.

Our data in non-neuronal cells are also reminiscent of those obtained in PIP5KI{gamma} knockout nerve terminals, where defects in the endocytosis as well as in the recycling of synaptic vesicles were observed. Thus, PtdIns[4,5]P2 appears to be involved in several trafficking steps in the endocytic pathway, regardless of cell types. Future directions will address how interactions of PtdIns[4,5]P2 with distinct binding proteins are coordinated to perform a sequential series of vesicular trafficking events.

ACKNOWLEDGMENTS

This research was supported by a grant of the Korea Health 21 R&D Project (03-PJ1-PG3–21300–0045) to S. Chang from Ministry of Health and Welfare, Republic of Korea. G. Di Paolo is funded by a grant from the National Institute of Health (HD047733–01).

Received for publication August 13, 2005. Accepted for publication June 6, 2006.

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