OCRL controls trafficking through early endosomes via PtdIns4,5P₂-dependent regulation of endosomal actin - PubMed (original) (raw)

. 2011 Oct 4;30(24):4970-85.

doi: 10.1038/emboj.2011.354.

Antonella Di Campli, Elena Polishchuk, Michele Santoro, Giuseppe Di Tullio, Anna Godi, Elena Levtchenko, Maria Giovanna De Leo, Roman Polishchuk, Lisette Sandoval, Maria-Paz Marzolo, Maria Antonietta De Matteis

Affiliations

OCRL controls trafficking through early endosomes via PtdIns4,5P₂-dependent regulation of endosomal actin

Mariella Vicinanza et al. EMBO J. 2011.

Abstract

Mutations in the phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P(2)) 5-phosphatase OCRL cause Lowe syndrome, which is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction. Previous studies have shown that OCRL interacts with components of the endosomal machinery; however, its role in endocytosis, and thus the pathogenic mechanisms of Lowe syndrome, have remained elusive. Here, we show that via its 5-phosphatase activity, OCRL controls early endosome (EE) function. OCRL depletion impairs the recycling of multiple classes of receptors, including megalin (which mediates protein reabsorption in the kidney) that are retained in engorged EEs. These trafficking defects are caused by ectopic accumulation of PtdIns4,5P(2) in EEs, which in turn induces an N-WASP-dependent increase in endosomal F-actin. Our data provide a molecular explanation for renal proximal tubular dysfunction in Lowe syndrome and highlight that tight control of PtdIns4,5P(2) and F-actin at the EEs is essential for exporting cargoes that transit this compartment.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

OCRL associates with megalin-containing endosomes. (A) HK2 cells expressing the HA–megalin (HA-Meg4) mini-receptor at steady state were stained for megalin (green) and OCRL (red), as indicated. OCRL and megalin partially colocalised in endosomal perinuclear structures (28% of megalin-containing structures were positive for OCRL). Inset: detail of boxed area. (B) HK2 cells expressing HA–Meg4 were initially kept for 2 h in serum-free medium, then incubated with a mouse anti-HA monoclonal antibody on ice for 30 min (to label the PM pool of HA–Meg4). They were then warmed to 37°C for 5 and 20 min (_t_5 and _t_20), given an acid wash, fixed, and incubated with an anti-OCRL polyclonal antibody and with secondary anti-mouse and anti-rabbit antisera. Insets: enlargement of the boxed area, with merged images of HA–Meg4 (green) and OCRL (red). After 5 min at 37°C, about 30% of the megalin-positive puncta contained OCRL. After 20 min, 78% of the perinuclear structures that contained megalin also contained OCRL. (C) HK2 cells were treated with non-targeting siRNA (CTR) or OCRL siRNAs (OCRL KD) for 96 h, transfected with HA–Meg4 for the last 18 h, and double labelled with antibodies against HA (upper panels) and against EEA1 or APPL1 (lower panels) as indicated. Insets: merged images of the boxed area, with megalin (green) and endocytic markers (red). Scale bar, 10 μm.

Figure 2

Figure 2

OCRL is required for megalin recycling to the PM. (A) HK2 cells were treated with non-targeting siRNA (CTR) or OCRL siRNAs (OCRL KD) for 96 h and transiently transfected with HA–Meg4 for the last 18 h. The PM exposure of HA–Meg4 and its internalisation were measured through binding at 4°C (cell surface HA–Meg4) and internalisation at 37°C for 5 min (internalised anti-HA Ab) of an anti-HA monoclonal antibody. The total amount of HA–Meg4 expressed was measured using an anti-HA polyclonal antibody (total HA–Meg4) in permeabilised HA–Meg4 cells. (B) Quantitative analysis of cell surface HA-Meg4 measured in cells exposed to the anti-HA monoclonal antibody for 1 h at 4°C (left graph), internalisation of HA–Meg4 measured at 5 and 20 min as uptake of anti-HA monoclonal antibody by HA–Meg4 HK2 cells treated as described in (A) (middle graph). Right graph: ratios of internalised/bound anti-HA monoclonal antibody at 5 and 20 min. All of the fluorescence intensities of the anti-HA monoclonal antibody (either at the cell surface or internalised) are normalised for total HA–Meg4 content (measured as described in (A), and expressed as the ratio between the mean fluorescence intensity of the anti-HA monoclonal antibody and that of the anti-HA polyclonal antibody). (C) HA–Meg4 recycling: HA–Meg4 HK2 cells were loaded with the anti-HA monoclonal antibody at 37°C for 30 min (LOAD) and then chased in fresh medium for 20 and 40 min at 37°C, and acid washed (CHASE). Data are mean values±s.d. (_n_=100 cells; three independent experiments). *P<0.01 and **P<0.001. (D, E) Uptake of GST–RAP in control PTCs (CTR; D, E), Lowe PTCs (D, E), and OCRL-KD PTCs, HK2, and MDCK cells (E) (by siRNA treatment), as indicated. RAP internalisation was quantified in (E) as the amount of cell-associated fluorescence, and expressed as % CTR. Scale bar, 10 μm. Data are mean values±s.d. (_n_=100 cells; three independent experiments).

Figure 3

Figure 3

OCRL KD impairs recycling of the TfR. (A) Control (CTR) or OCRL-KD HeLa cells were exposed to Alexa-Fluor-488 (A488)-Tf for 1 h at 4°C and then warmed to 37°C in complete medium for 5 min. Scale bar, 10 μm. (B) Quantification of cell-associated A488-Tf, evaluated as mean fluorescence intensities at indicated times, and expressed as indicated. (C) For Tf recycling, the cells were loaded with Alexa-Fluor-488-Tf for 1 h at 37°C (LOAD) and chased in complete medium for 40 and 60 min (CHASE). The fluorescence intensities remaining in the cells after 40 and 60 min of chase were quantified and expressed as percentages of the loaded Tf. Data are mean values±s.d. (_n_=150 cells; three independent experiments). (D) Steady-state distributions of the TfR and MPR in CTR and OCRL-KD HeLa cells. Insets: enlargement of the boxed area, with merged images of TfR (green) and MPR (red). Scale bar, 10 μm. (E) Steady-state localisation of the TfR in CTR and OCRL siRNA-treated HeLa cells was visualised by pre-embedding immuno-gold labelling with an anti-TfR antibody. E, endosomes; G, Golgi complex; PM, plasma membrane. Scale bar, 100 nm.

Figure 4

Figure 4

OCRL KD impairs recycling of the MPR to the PM and the Golgi complex. (A) Uptake of human α-glycosidase (GAA) was determined in control (CTR) and OCRL-KD HeLa cells incubated with Alexa-Fluor-546 (A546)-recombinant human GAA (A546-GAA) for 2 h at 37°C. White lines, approximate cell contours. The amount of internalised A546-hGAA (quantified by fluorescence intensity) decreased by 50% (±5%) of the CTR in the OCRL-KD cells (_n_=80 cells; three independent experiments). (B) PM exposure of the MPR and its internalisation were measured through binding at 4°C (1 h 4°C) and internalisation for 15 min at 37°C (+15 min 37°C) of an anti-MPR antibody directed against the luminal epitope of MPR (anti-MPR Ab) in control (CTR) and OCRL-KD HeLa cells. After fixation and treatment with the Alexa-568 secondary antibody, the cell-associated fluorescence was quantified and expressed as %CTR. (C, D) Steady-state distributions of the MPR and TfR in PTCs from healthy subjects (CTR) and Lowe syndrome patients (Lowe). In (C), the insets show enlargements of the boxed areas, with merged images of the TfR (green) and the MPR (red). (D) Images from control PTCs (CTR), Lowe PTCs, and PTCs from healthy subjects knock down for OCRL via siRNA treatment (OCRL KD) labelled for the MPR, as acquired and quantified by Scan^R automated microscopy (see Supplementary data). The percentages of cells showing the different MPR staining patterns were classified as indicated; these are mean values±s.d. (_n_=1000 cells; three independent experiments, each performed in quadruplicate). *P<0.01 and **P<0.001. Scale bar, 10 μm. (E) Colocalisation of the MPR with Rab proteins in control (CTR) and OCRL-KD HeLa cells transiently expressing GFP-tagged Rab4 and Rab5. Insets: enlargements of boxed areas, with merged images of Rab4 or Rab5 (green) and the MPR (red). Scale bar, 10 μm.

Figure 5

Figure 5

OCRL regulates the phosphoinositide composition of early endosomes. (A) OCRL-KD HeLa cells (left panel) and those injected with cDNA encoding for siRNA-resistant GFP-tagged wild-type (OCRL wt, middle panels) or catalytically inactive (OCRL V527D, right panels) OCRL were stained for the MPR (red). (B–D) The distribution of MPR (B), uptake of Alexa 568-Tf (C), and uptake of the anti-HA antibody (D) were evaluated and quantified in HeLa cells (B, C), and HA–Meg4 HK2 cells (D), which were knocked down for OCRL and transfected with siRNA-resistant GFP-tagged wild-type (OCRL wt) or catalytically inactive (OCRL V527D) OCRL for the last 6 h. HeLa cells were exposed to Alexa 568-Tf for 1 h at 37°C and HK2 cells were exposed to the anti-HA antibody for 30 min at 37°C. The data are expressed as % analysed cells with peripheral redistribution of the MPR in (B) or as % control cells (cells which were neither transfected with GFP–OCRL nor treated with siRNA for OCRL) in (C) and (D). The data in each graph refer to at least three independent experiments. (E–G) Subcellular localisation of PtdIns4,5P2 in CTR and OCRL-KD cells determined using the PH-PLCδ PtdIns4,5P2 probe. (E) GFP–PLCδ-PH-expressing cells were exposed to 50 μg/ml Alexa-Fluor-568 (A568)-Tf for 30 min, rinsed in complete medium, and imaged at 37°C for 40 min. Still images from a time-lapse movie are shown. PLCδ-PH-negative and PLCδ-PH-positive Tf-containing structures are indicated in the boxed areas by empty and filled arrowheads, respectively. As the OCRL-KD cells take up less Tf compared with mock cells, the setting of the A568-Tf channel was adjusted (with higher laser power and detector amplifier) to visualise an adequate number of Tf-containing structures in these cells. (F) CTR and OCRL-KD cells were processed for immuno-gold labelling with GST–PH-PLCδ (10 nm gold particles) and an anti-TfR antibody (15 nm gold particles). E, endosomes. Note that some of the endosome intralumenal vesicles (ILVs) in OCRL-KD cells are positive for GST–PH-PLCδ. (G) Morphometric analysis (performed as described in Materials and methods) of the labelling density of GST–PH-PLCδ in different cellular compartments in CTR cells and in OCRL-KD cells. The data are expressed as mean±s.d.; for PM, _n_=43 (CTR) and 48 (OCRL KD) cells; for endosomes, _n_=61 (CTR) and 82 (OCRL KD) endosomes; for Golgi, _n_=28 (CTR) and 24 (OCRL KD) stacks. Scale bars, 10 μm (A, E) and 100 nm (F).

Figure 6

Figure 6

OCRL KD induces an increase in F-actin on early endosomes, which has a causative role in early endosome dysfunction. (A) Control (CTR) and OCRL-KD HK2 cells were stained with Alexa-Fluor-488 (A488)-phalloidin (green) to visualise F-actin and with anti-EEA1 antibody (red) to visualise EE compartments. (B) Control (CTR) and OCRL-KD HK2 cells expressing HA–mini-megalin were incubated with an anti-HA antibody at 37°C for 30 min, then fixed and stained with Alexa-Fluor-488 (A488)-phalloidin (green) to visualise F-actin, and with secondary antibody directed against the anti-HA antibody (red) to visualise internalised megalin. (C) Control (CTR) and OCRL-KD HK2 cells were stained with anti-p16Arc antibodies to visualise the Arp2/3 complex, and with anti-EEA1 antibodies to visualise EE. Insets show enlargements of the boxed areas, with merged images of p16-Arc (green) and EEA1 (red). (D) Control cells and OCRL-KD cells were either left untreated (none) or were treated with latrunculin B (LatB) at the indicated concentrations or transfected with wild-type (wt) cofilin or a constitutively active non-phosphorylatable mutant of cofilin (S3A) 1 day before the experiment, or treated with siRNAs directed against N-WASP (N-WASP-KD) or WASH (WASH KD) for 3 days before the experiments. The cells were then either stained for F-actin (with phalloidin) or for the MPR, or were exposed to Tf (1 h at 37°C, HeLa cells) or to anti-HA antibody (1 h at 37°C, HA–megalin-expressing HK2 cells). The amounts of F-actin associated with endosomes loaded with Tf were analysed as described in Materials and methods and expressed as % endosomes positive for F-actin±s.d. Uptake of Tf and of the anti-HA antibody and the number of cells with a central distribution of MPR were analysed and quantified as described in Materials and methods and are expressed as % CTR cells (i.e., untreated cells). *P<0.01 and **P<0.001.

Figure 7

Figure 7

Knockdown of PIP 5-kinases rescues the endosomal trafficking defects and the F-actin changes in OCRL-depleted cells. (A) Internalised Alexa-Fluor-488 (A488)-Tf (5 min at 37°C) was visualised and quantified in control cells (CTR), OCRL-KD cells (OCRL KD), PIP5Kα-KD cells, PIP5Kβ-KD cells, PIP5Kγ-KD, and OCRL-KD cells upon siRNA-mediated depletion of the different PIP5K isoforms (OCRL+PIP5Kα KD, OCRL+PIP5Kβ KD, and OCRL+PIP5Kγ KD). (B) MPR distribution, GST–RAP uptake (5 min at 37°C), F-actin and Arp2/3 complex associated with Tf-loaded endosomes, expressed as indicated, were quantified in CTR cells, OCRL-KD cells, PIP5Kα-KD cells, and in OCRL-KD cells upon siRNA-mediated depletion of PIP5Kα (OCRL+PIP5Kα KD). The data are mean values±s.d. (_n_=100 cells for Tf and GST–RAP uptake, MPR distribution; _n_=10 cells for F-actin and Arp2/3 endosomal localisation; from three independent experiments); *P<0.01 and **P<0.001. (C) Model of the role of OCRL in the early endocytic trafficking pathway. Left panel: Snj2 and brain-specific, 145 kDa Snj1 are recruited at the early and late steps, respectively, of clathrin-coated pit (CCP) and clathrin-coated vesicles (CCVs) formation; the ubiquitously expressed 170 kDa SJ1 is present throughout CCP and CCV formation. OCRL associates with CCV just before the release of clathrin and with early endosomes. Consumption of PtdIns4,5P2 and generation of PtdIns3P via type III PI3K are under the control of the GTPase Rab5. Right panel: impact of OCRL loss of function on endosomal PtdIns4,5P2 and F-actin levels and, as a consequence, on recycling from early endosomes (see text for detail).

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