FCHo proteins are nucleators of clathrin-mediated endocytosis

Abstract

Clathrin-mediated endocytosis, the major pathway for ligand internalization into eukaryotic cells, is thought to be initiated by the clustering of clathrin and adaptors around receptors destined for internalization. However, here we report that the membrane-sculpting F-BAR domain-containing Fer/Cip4 homology domain-only proteins 1 and 2 (FCHo1/2) were required for plasma membrane clathrin-coated vesicle (CCV) budding and marked sites of CCV formation. Changes in FCHo1/2 expression levels correlated directly with numbers of CCV budding events, ligand endocytosis, and synaptic vesicle marker recycling. FCHo1/2 proteins bound specifically to the plasma membrane and recruited the scaffold proteins eps15 and intersectin, which in turn engaged the adaptor complex AP2. The FCHo F-BAR membrane-bending activity was required, leading to the proposal that FCHo1/2 sculpt the initial bud site and recruit the clathrin machinery for CCV formation.

Fig. 1

FCHo1/2 proteins are clathrin/AP2 nucleators. (A) Dynamic cell surface localization (top) and kymograph (bottom) of representative CCPs labelled with RFP-FCHo2 (FCHo2) and GFP-LCa (clathrin). FCHo2 was detected before clathrin (white arrows and graph). a.u.: arbitary units. (B) Cryoimmuno-electron microscopy localized GFP-FCHo2 at CCPs. Scale bar 100nm. (C) CCVs, labelled by σ2-GFP (AP2), did not form with double RNAi of FCHo1+2 (FCHo1+2 RNAi) where AP2 became cytosolic (contrast enhanced to show the diffuse signal at the plasma membrane). Inhibition was relieved by co-expression of RNAi-resistant RFP-FCHo2 (rescue). (D) Nucleation rates (number of new CCPs/10⁴μm²/second) in cells treated with scrambled RNAi (Ctrl), RNAi against FCHo1 (1), FCHo2 (2), FCHo1+2 (1+2) or rescue (R). (E) Clathrin ligands, transferrin (Tf) epidermal growth factor (EGF) and low-density lipoprotein (LDL) uptake in cells treated as in C. (F) Clathrin vesicles (AP2) in BSC1 cells transfected with 0, 1 or 2μg of untagged-FCHo2 for 2×10⁵ cells. (G) Nucleation rate and (H) Tf uptake in cells treated as in F. Scale bars, 5μm (C,F) and 200nm (B). Displayed kymographs were representative (percentage, n=319 CCPs (11)).

Fig. 2

FCHo2 directly binds and recruits eps15 and intersectin to initiate CCP maturation. (A) Pull down with GST-FCHo2-μHD and rat brain lysate. Interacting proteins were identified by mass spectrometry. (B) Eps15 and intersectin (ITSN) formed puncta at the PM colocalizing with FCHo2. In double FCHo1+2 RNAi cells, Eps15 and ITSN were cytosolic (contrast enhanced to show the diffuse signal); co-expression of RNAi-resistant FCHo2 (FCHo2r) rescued PM-targeting (Rescue). (C) Kymograph of representative CCPs (percentage, (11)) labeled with FCHo2 and Eps15 or ITSN. (D) FCHo2, eps15, and ITSN were CCV de-enriched. Clathrin (CHC), AP2, and vesicle marker synaptophysin (Syphy) displayed enrichment in CCV fractions (CCV). FCHo2 and ITSN were PM enriched. IB: immunoblot. (E) Cryoimmuno-electron microscopy of GFP-FCHo2 localized it to the CCP neck. Bar graph shows gold particle density in the upper and lower half of constricted CCPs (p<0.01). (F) Pull downs with GST-μHD from scrambled (left) or AP2 RNAi (right) treated HeLa cells. Eps15 and ITSN bands were visible in both (arrows). (G) Upon AP2 depletion (μ2 RNAi) σ2-GFP (another AP2 subunit) was cytosolic (contrast enhanced to show the diffuse signal), but Eps15 and ITSN still co-localize with FCHo2 at the PM (arrows). (H) FCHo2 and AP2 (σ2-GFP) puncta disappeared under Eps15 + Eps15R + ITSN1 + ITSN2 quadruple RNAi. AP2 became cytosolic (diffuse signal) whereas FCHo2 remained at the PM (inset). (I) In FCHo1+2 RNAi cells, RNAi-resistant FCHo2-K797E (FCHo2r-K797E) bound to the PM (arrows) but did not cluster nor rescue CCP formation, reported by RFP-LCa (clathrin). In these cells FCHo1+2 RNAi inhibition of Tf uptake was also not rescued (7.2±3.5% of control uptake (p<0.0001). (J) GST-μHD K797E no longer pulled-down the protein bands visible in A. Green, red and blue panels indicate GFP-, RFP-, and BFP-tagged proteins respectively. Scale bars, 5μm. (B,H,G,I) and 100nm (E).

Fig. 3

Lipid binding and membrane sculpting FCHo1/2 abilities are both essential for CCP formation. (A) Chimeric GFP-PLC-PH+FCHo2μHD (PH-μHD), a dimer interface mutant GFP-FCHo2(F38E+W73E), and RFP-SGIP1 all could not rescue CCP nucleation - monitored by following either clathrin or AP2 fluorescence - in double RNAi FCHo1+2 cells. (B) Lipid co-sedimentation assay of 15μM F-BAR-x in presence of 1mg/mL liposomes: Folch (Avanti brain lipid), FolchPS (80% Folch, 20% phosphatidylserine) or FolchPS +5% of indicated PiPs. Liposome-bound proteins were pelleted (P) by ultracentrifugation, unbound protein remained in the supernatant (S). (C) Folch+15%PS+5%Pi(4,5)P₂ liposomes incubated with either 10 or 20μM F-BAR-x and spotted onto EM grids gave mainly tubules of diameters of 50-80nm (10 μM) or 18nm (20μM, most were visibly twisted). Insets show enlargements with protein density striations. (D) F-BAR-x-induced in vivo tubulation: representative images of ‘tubules’ and ‘no tubules’. (E) RNAi-resistant form of full-length FCHo2 (FCHo2r) with membrane-binding mutation (K146E+K163E) remained cyosolic and could not rescue FCHo1/2 RNAi-mediated absence of CCPs, whereas FCHo2r containing membrane-sculpting mutations (I268N and L136E) displayed slowed and aberrant CCPs. Displayed kymographs were representative (percentage, (11)). Table summarizing the mutants and their phenotypes. Scale bars, 5μm (A,D,E) and 100nm (C).