ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling

The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE.

. Endosome size and distribution are not significantly altered in Alix ko cells. a) wt and Alix ko MEFs were immunolabelled to reveal specific endosomal compartments using the endosomal markers as follows: EEA1 (early endosomes), Rab 11 (recycling endosomes), LAMP1 (late endosomes and lysosomes) and LBPA (multivesicular bodies). wt and Alix ko MEFs were also labelled with filipin to stain       were incubated with CTxB for 20 min at 4°C, followed by 5 min at 37°C and internalized CTxB was quantified. Cells were incubated with filipin to disrupt CIE or chlorpromazine (chlorpro) to disrupt CME, prior to and during CTxB internalization (Number of cells in

Supplementary Videos legends:
Supplementary Video1 : Localization of GFP-Alix in resting condition.
Alix ko MEF cells expressing GFP-Alix (green) were observed by time-lapse confocal microscopy using a spinning disk confocal microscope (AxioObserver Z1, Zeiss). Frames were taken every 2 secondes during 1 minute.
Alix ko MEF cells expressing GFP-Alix (green) were incubated with 2mg/ml CTxB-TRITC (red) 5 min prior to the beginning of the video. Images were acquired as in video during 5 minutes.

Supplementary material Cholesterol and LBPA quantifications
The analysis of cholesterol distribution was done by fluorescence microscopy after fixation and treatment of cells with 50 g/ml filipin. For LBPA, 6C4 antibody was used for immunolabeling. Samples were imaged with a confocal microscope using a 40x oil immersion objective. Mean fluorescence intensity was measured using ImageJ and results are expressed as a fraction of wt uptake considered as 1.

Cell spreading assay
MEFs were trypsinized, replated on coverslips precoated with 10 g/ml fibronectin (FN), and allowed to spread for 2 h at 37°C in complete medium. Cultures were fixed and visualized by staining with phalloidin-TRITC for 1 h and the mean surface area was calculated using ImageJ.

GPI-GFP recycling
GPI-GFP recycling was estimated using the method described in 1 . Briefly, GPI-GFP transfected MEFs were rinsed with cold DMEM containing 0.2% BSA and 20 mM HEPES and incubated for 45 min on ice with monoclonal anti-GFP (4 g/ml). Cells were then incubated at 37°C for 10 min to allow endocytosis. Surface bound antibodies were then removed by a 2 min incubation with 25 mM sodium acetate in DMEM (pH 2) followed by neutralization with 25 mM Tris in DMEM, pH 10. Cells were then brought back to 10 min at 37°C and immunostained after fixation with anti-GFP antibodies to estimate the amount of GPI-GFP which had reappeared at the cell surface. This recycled GPI-GFP pool is then compared to the total pool of GPI-GFP, estimated by immunostaining of cells after permeabilization using 0.2% saponin. The samples were imaged using a Zeiss Axiovert 200 microscope equipped with a 20x or 40x objective and the mean fluorescence intensity was measured using ImageJ. Intensities were normalized to binding of anti-GFP antibody at 4°C. Results are expressed as a fraction of uptake by wt cells considered as 1.

Localization of CTxB, TfR and Alix in Detergent-Resistant Membrane Domains (DRMs)
For localization of Alix in DRMs, Alix ko MEFs transfected with GFP-Alix were labelled with CTxB-TRITC or TfR-TRITC for 30 min at 4°C, washed with cold PBS and treated with 0.5% Triton X-100 for 1 min to remove the detergent-sensitive membrane fraction.
Cells were then washed with PBS, fixed with 4% PFA and analysed with the Zeiss LSM 710 inverted confocal microscope.
Live Imaging of GFP-Alix recruitment to CTxB entry sites