Endocytosis mediates the cellular uptake of micronutrients and the turnover of plasma membrane proteins. Clathrin-mediated endocytosis is the major uptake pathway in resting cells1, but several clathrin-independent endocytic routes exist in parallel2,3. One such pathway, fast endophilin-mediated endocytosis (FEME), is not constitutive but triggered upon activation of certain receptors, including the β1 adrenergic receptor4. FEME activates promptly following stimulation as endophilin is pre-enriched by the phosphatidylinositol-3,4-bisphosphate-binding protein lamellipodin4,5. However, in the absence of stimulation, endophilin foci abort and disassemble after a few seconds. Looking for additional proteins involved in FEME, we found that 20 out of 65 BAR domain-containing proteins tested colocalized with endophilin spots. Among them, FBP17 and CIP4 prime the membrane of resting cells for FEME by recruiting the 5′-lipid phosphatase SHIP2 and lamellipodin to mediate the local production of phosphatidylinositol-3,4-bisphosphate and endophilin pre-enrichment. Membrane-bound GTP-loaded Cdc42 recruits FBP17 and CIP4, before being locally deactivated by RICH1 and SH3BP1 GTPase-activating proteins. This generates the transient assembly and disassembly of endophilin spots, which lasts 5–10 seconds. This mechanism periodically primes patches of the membrane for prompt responses upon FEME activation.
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We thank S. Y. Peak-Chew for mass spectrometry, M. Edwards, M. Dumoux and K. McGourty for technical help, P. De Camilli (Yale University), P. Aspenstrom (Karolinska Institute), P. Randazzo (NIH), M. Negishi (Kyoto University), G. Rappold (University of Heidelberg), H. Kent (MRC Laboratory of Molecular Biology), J. Gallop (University of Cambridge), T. Takeda (MRC Molecular Biology), M. Parsons (King’s College London), F. Gertler (MIT) and T. Kirchhausen (Harvard Medical School) for the kind gift of reagents and the members of the Boucrot lab for helpful comments. E.B. was a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Research Fellow (BB/R01551X/1), a Lister Institute Research Fellow and a recipient of a grant from the Royal Society Research Grant (RG120481). S.L.-A.H. and H.T.M. were supported by the MRC UK (grant number U105178805 to H.T.M.) and by the Swiss National Science Foundation (fellowship number PA00P3-124164 to S.L.-A.H). A.P.A.F was supported by the Fundação para a Ciência e Tecnologia.
The authors declare no competing interests.
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Integrated supplementary information
a, Pull down using GST-SH3 domains for Endophilin A1, A2 or A3 and rat brain lysate. Interacting proteins were identified by mass spectrometry. Known interactors are shown in grey, members of the WAVE complex in black and BAR-domain proteins in blue. Asterisk denotes GST-SH3. List of identified proteins is provided in Supplementary Table 1. b, Pull-down using GST-SH3 domains of Endophilin A2 and cell extracts expressing EGFP-tagged indicated BAR proteins. Bin2 and OPHN1 were used as positive controls. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. ‘input’ lanes correspond to 1-5 % of the cell extracts. c, Schematic depicting the domain organization of the human full-length BAR domain-containing proteins cloned and used in this study. d, co-immunoprecipitation assays from cells co-expressing the indicated combination of EGFP- or Myc-tagged BAR domains from FBP17, CIP4 and TOCA-1 and showing that all three can heterodimerize. Input (‘I’, which corresponds to 10% of cell extracts), unbound (‘U’) and bound (‘B’) fractions for each were immunoblotted with anti-EGFP and anti-Myc antibodies. All experiments were repeated independently at least three times with similar results. Unprocessed original scans are provided in Supplementary Fig. 6.
a, Number of tissues where expressed sequence tag (EST) were detected in the UniGene transcriptome database for the named human BAR domain-containing proteins. Zones for expression in 25% and 50% of the tissues are highlighted in dark and light red, respectively. b, Number of transcript per million (TPM) in kidney (tissue of origin of BSC1 cells) for the named human BAR domain-containing proteins recorded in the UniGene transcriptome database. Red boxes highlights genes for which no transcript were detected. c, Number of transcript per million (TPM) in eye (tissue of origin of RPE1 cells) for the named human BAR domain-containing proteins recorded in the UniGene transcriptome database. Red boxes highlights genes for which no transcript were detected. The data were recorded from the NCBI UniGene transcriptome database (see Methods for details).
a, Confocal microscopy sections showing an example of the named BAR proteins tagged with EGFP at their C-termini colocalizing (RICH1-EGFP, left) or not (RICH2-EGFP, right) with endogenous Endophilin in BSC1 cells. b, Criteria used to score BAR candidates negative or positive. Intensity profiles were acquired along the indicated lines. c-f, Confocal microscopy sections showing the colocalization of the named controls (soluble EGFP and CAAX-EGFP), BAR, N-BAR or BAR-PH proteins tagged with EGFP at their C-termini and Endophilin. Images were cropped from larger pictures and angled as in (a) so that all images are oriented similarly. Arrowheads point to Endophilin spots colocalizing with BAR proteins at the cell surface. Images are representative of 10 captures from three independent biological experiments. All experiments were repeated independently at least three times with similar results. Scale bars, 5 μm.
a-c, Confocal microscopy sections showing the colocalization of the named PX-BAR, F-BAR or I-BAR proteins tagged with EGFP at their C-termini and Endophilin in BSC1 cells. Arrowheads point to Endophilin spots colocalizing with BAR proteins at the cell surface. Images are representative of 10 captures from three independent biological experiments. d, Absence of colocalization between internalized Alexa488-Transferrin and endogenous Endophilin in BSC1 and RPE1 cells stimulated with 10μM dobutamine and 50μg/mL Alexa488-Transferrin for 30sec at 37 °C for 30sec. Images are representative of 10 captures from three independent biological experiments. e, Top, β1-AR and GAPDH levels in the indicated plasma membrane and total fractions from BSC1 cells depleted or not for Endophilin (Endo TKD), AP2, FBP17 and CIP4 (F+C DKD) or Amphiphysin and Bin1 (A+B DKD). Unprocessed original scans are provided in Supplementary Fig. 6. f, Colocalization of β1-adrenergic receptor (β1-AR) and LAMP1 in resting BSC1 cells or upon incubation with 10μM dobutamine for 30min. Images are representative of 6 captures from three independent biological experiments. All experiments were repeated independently at least three times with similar results. Scale bars, 20 (f) and 5μm (a,,b,c,d).
a, Confocal microscopy sections showing the transient increase in endogenous CIP4 and Endophilin on FEME carriers after 2 but not 10min incubation with 10μM ML141 (‘Cdc42i 1’). Images are representative of 6 captures from three independent biological experiments. b, Confocal microscopy section showing the decrease or not in endogenous Endophilin recruitment at the leading edge of resting cells overexpressing wild-type EGFP-tagged RICH2, SH3BP1 or OPHN1 but not their R291A, R312A and R409A respective GAP mutants (GAP*). Arrowheads point to Endophilin spots at the cell surface. Images are representative of 6 captures from three independent biological experiments. c,d, Recruitment of endogenous RICH1, Lpd and CIP4 in resting BSC1 cells depleted or not for Lamellipodin (Lpd KD), RICH1 and SH3BP1 (R+S DKD) or FBP17 and CIP4 (F+C DKD). Images are representative of 10 captures from three independent biological experiments. e, Pull-down using GST or GST-SH3 domains of Endophilin A2 or FBP17 and cell extracts expressing RICH1, RICH2, SH3BP1 or OPHN1 tagged with EGFP at their C-termini. Inputs correspond to 1 to 5% of the cell extracts. All experiments were repeated independently at least three times with similar results. Unprocessed original scans are provided in Supplementary Fig. 6. Bars, 5 μm.
Unprocessed original scans of all gels and blots.
Supplementary Figures 1–6, Supplementary Table and Supplementary Video legends.
List of interactors identified in Supplementary Fig. 1a.
Statistics Source Data.
Sequences of DNA and siRNA primers used in this study.
Information on antibodies used in this study.
Spinning-disk confocal microscopy of a resting BSC1 cell transiently expressing low levels of EGFP–LCa (clathrin, green) and endophilin A2-RFP (red) and imaged at 0.5 Hz.
Spinning-disk confocal microscopy of a resting BSC1 cell transiently expressing low levels of FBP17–EGFP (green) and Endophilin A2–RFP (red) and imaged at 0.5 Hz.
Spinning-disk confocal microscopy of a resting BSC1 cell transiently expressing low levels of CIP4–EGFP (green) and Endophilin A2–RFP (red) and imaged at 2 Hz. 5 nM GDC-0941 (PI3Ki) was added at timeframe 100.
Spinning-disk confocal microscopy of a resting BSC1 cell transiently expressing low levels of FBP17–EGFP (green) and Endophilin A2–RFP (red) and imaged at 2 Hz.
Spinning-disk confocal microscopy of a resting BSC1 cell transiently expressing low levels of CIP4–EGFP (green) and Endophilin A2–RFP (red) and imaged at 2 Hz.
Spinning-disk confocal microscopy of a resting BSC1 cell transiently expressing low levels of CIP4–EGFP (green) and Endophilin A2–RFP (red) and imaged at 2 Hz. 5 μM AS19499490 (‘SHIP2i’) was added at timeframe 100 and again at timeframe 200.
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Chan Wah Hak, L., Khan, S., Di Meglio, I. et al. FBP17 and CIP4 recruit SHIP2 and lamellipodin to prime the plasma membrane for fast endophilin-mediated endocytosis. Nat Cell Biol 20, 1023–1031 (2018). https://doi.org/10.1038/s41556-018-0146-8
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