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

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

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

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

SPECIFIC AIMS

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P₂), a phosphoinositide involved in a variety of cellular functions, appears to play a general role in membrane trafficking including early endocytosis, although the precise trafficking steps relying on normal PtdIns[4,5]P₂ balance in the endosomal pathway have not been elucidated yet. Therefore, this study was conducted to get further insight on the role of PtdIns[4,5]P₂ in transferrin recycling kinetics.

PRINCIPAL FINDINGS

1. Overexpression of 5-phosphatase domain of synaptojanin decreased PtdIns[4,5]P₂ levels
As a prelude to our functional studies, we tested whether the overexpression of the inositol 5-phosphatase domain of synaptojanin 1 (5-PPase) in COS-7 cells could significantly affect PtdIns[4,5]P₂ metabolism. There is a reciprocal relation between PtdIns[4,5]P₂ and PtdIns[4]P, and the extract from cells overexpressing 5-PPase decreased PtdIns[4,5]P₂ substrate levels 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%). We further showed that the coexpression of PtdIns[4]P 5-kinase type I (PIP5KI) with 5-PPase restores normal PtdIns[4,5]P₂ metabolism (control vs. coexpression; PtdIns[4,5]P₂: 49.7 vs. 41.7%; PtdIns[4]P: 50.3 vs. 58.3%).

2. Transferrin endocytosis and recycling are impaired by the decrease of PtdIns[4,5]P₂ levels
To test the role of PtdIns[4,5]P₂ in endocytosis, we first performed a transferrin uptake assay in COS-7 cells that were transfected with 5-PPase, the PH domain of PLC (PLC-PH), or the PtdIns[4,5]P₂ binding peptide of gelsolin (PBP). In nontransfected cells, Texas red transferrin was internalized and accumulated in the perinuclear endosomal compartment and in punctate structures dispersed throughout the cytoplasm, while in cells overexpressing 5-PPase, PLC--PH or PBP, no such accumulation was observed (average Texas red transferrin fluorescence in a.f.u: 5-PPase, 54.0±12.4; PLC-PH, 78.3±8.1; PBP, 88.4±8.7; control, 173.4±7.0). Coexpression of PIP5KI with 5-PPase restored transferrin uptake and endocytosis to a level comparable with 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).

To test whether PtdIns[4,5]P₂ could affect more than one trafficking step of transferrin cycle, we performed a single round of transferrin uptake and recycling assay. In control cells, transferrin was endocytosed rapidly within 5 min, and the internalized transferrin was efficiently recycled out to the plasma membrane. In contrast, cells overexpressing 5-PPase showed significant recycling defects as well as endocytosis defect, whereby the initial rate of transferrin uptake decreased 2-fold relative to the control (Fig. 1 A, inset), and a significant fraction of the internalized transferrin still remained in the cells >60 min after uptake in contrast to control cells, which <20% remained (Fig. 1A ).

To rule out the possibility in that the delay in transferrin recycling in cells overexpressing 5-PPase could be simply due to the initial delay in transferrin internalization, cells were incubated with Texas red transferrin for 60 min at 37°C to saturate the entire recycling pathway. Figure 1B 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. 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. 1B ). When we compared the first 10 min of recycling kinetics (Fig. 1B , inset), control cells released more than half of the internalized Texas red transferrin while cells overexpressing 5-PPase retained most of internalized Texas red transferrin (% of initial fluorescence remained: 44.9 vs. 91.8%). After 10 min of lag phase, cells overexpressing 5-PPase started to release transferrin although at a slower rate compared with that of control. 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.

3. 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 that involves vesicular transport from the sorting endosome to the plasma membrane, in addition to the slow recycling pathway previously characterized, has been suggested. 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. Cells were first labeled with FM 5–95 for 1 min, and the efflux kinetics of FM 5–95 was imaged. 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. 2 ). In cells overexpressing 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. 2 ) and more importantly, they retained a higher fraction of residual FM 5–95 fluorescence after 60 min incubation (24.2% for control, 42.1% for 5-PPase), suggesting 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 cells overexpressing 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 a membrane recycling pathway.

CONCLUSIONS AND SIGNIFICANCE

In the current study, we have unmasked the role of PtdIns[4,5]P₂ in multiple trafficking and/or sorting events of transferrin in non-neuronal cells. Previous 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. 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. 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. 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 instances. 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. Finally, based on the known effects of PtdIns[4,5]P₂ on the actin cytoskeleton, 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 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.

View larger version (21K): [in this window] [in a new window]   Figure 1. Transferrin endocytosis and recycling are impaired by decrease of 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 the 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 accessed 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 the initial 10 min. Data are mean ± SD; n=5.

View larger version (27K): [in this window] [in a new window]   Figure 2. Membrane recycling is impaired by the decrease of 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 following a 1 min pulse were obtained by quantification of fluorescent images. Fluorescent values were normalized to that of the first value at the beginning of the chase. Data points fit well to a double exponential decay (R2>0.99), giving a half-time of 2 to 3 min for fast component and 20 to 25 min for slow component. Please note that after 60 min incubation, substantial amount of fluorescence was still retained in cells overexpressing 5-PPase. Data are mean ± SD; n = 4.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic figure of PtdIns[4,5]P2 involvement in transferrin and membrane recycling pathways. Transferrin recycles through a series of endosomal compartments. It is rapidly delivered to a sorting endosome and subsequently sorted to a recycling compartment, from which it returns to the plasma membrane. PtdIns[4,5]P2 can affect not only initial transferrin endocytic processes but also recycling of internalized transferrin back to cell surface. Lipids and other membrane constituents also recycle between plasma membrane and intracellular endocytic compartments. Fast recycling pathway involves vesicular transport from sorting endosome to plasma membrane, whereas slow pathway involves transport from recycling endosome. PtdIns[4,5]P2 plays a critical role after membrane internalization in one or multiple steps of a membrane recycling pathway. The involvement of PtdIns[4,5]P2 in exocytosis is still unclear.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4621fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-4621fjev1 20/13/2399    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, S. Articles by Chang, S. Search for Related Content PubMed PubMed Citation Articles by Kim, S. Articles by Chang, S.