genome editing of u-2 os cells to generate isogenic knockout clones of endogenous fcho2 (fcho2-ko)  (GenScript corporation)

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( A ) Cartoon representations of mammalian AP2 and muniscin family members FCHO1, FCHO2, and SGIP. AP2 is colored according to subunits as used throughout α (dark blue), β2 (dark green), Nμ2 (dark purple), Cμ2 (pale purple), and σ2 (cyan). Muniscins have FBAR and MHD domains joined by unstructured interdomain linkers. ( B ) Top: Side-view representation of a forming CCP imaged by eTIRF-SIM with an illumination depth of 100 nm (blue gradient). Clathrin is depicted either in green when visible or gray when not visible because of the exponentially decaying evanescent wave of TIRF. Bottom: 2D projection of clathrin (green) in a forming CCP along its axis of invagination into the plane of the PM. ( C ) eTIRF-SIM images of mScarlet-FCHO2 WT and egfp-CLCa in different phases of CCP formation. Region of interest (ROI) containing single CCPs in a time series was dissected into phases depending on the appearance of FCHO2 or egfp-CLCa. ROIs with individual CCPs in different phases were assembled into a z-stack and averaged across 150 CCPs in three different cells. The bottom row shows comparison of not normalized intensity distribution profiles (along the line indicated with white triangles) for CLC and FCHO2 (green and magenta, respectively) at different time points together with their full width at half maximum (FWHM). The black dotted line shows the measured point spread function (PSF). N/A, not applicable.

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques: Comparison

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( Left ) Averaged eTIRF-SIM images of multiple individual ROIs (as in C). The top row (green) shows CLC, and the middle row (magenta) shows pioneer proteins of interest (Eps15, AP2, and FCHO2). The bottom row demonstrates overlay between the channels highlighting the molecule distribution with respect to CLC. mScarlet-FCHO2 WT forms an annulus covering the outer rim of the clathrin hemispherical coat of averaged CLC (green line) that is 260 nm. ( Right ) Normalized egfp-CLCa intensity profiles along the line indicated with white triangles of the previous panel. The green solid line shows the CCP intensity profiles obtained from averaging images of 480 CCPs in U-2 OS cells with an estimated FWHM of 260 nm. While AP2 (α-adaptin egfp, shown in blue dashed line) forms a central patch in a mature CCP, Eps15 (polyclonal antibody, magenta dashed line) forms a transition zone with FWHM larger than that of FCHO2 (black) and egfp-CLCa (green). A.U., arbitrary units.

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques:

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( A ) Equilibrium analyses BLI showing similar binding of recombinant AP2 core to FCHO1 linker (residues 316 to 444) with K D of 8.5 ± 2.2 μM and to FCHO2 linker (residues 314 to 444) with K D of 9.8 ± 1.5 μM. ( B and C ) Coomassie-stained SDS–polyacrylamide gel electrophoresis (PAGE) gels of supernatant (S) and pelleted (P) fractions following addition of phosphatidylcholine (PC)/phosphatidylethanolamine (PE), PC/PE/PtdIns(4,5)P 2 , and PC/PE/PtdIns(4,5)P 2 +YxxΦ cargo liposomes to mixtures of glutathione S -transferase (GST)-FCHO2 linker (15 μM) and AP2 core (1.25 μM), which glutathione (GSH) Sepharose bead pull-downs show contain GST-FCHO2 linker/AP2 core complex (top) and free GST and AP2 core (bottom). AP2 core binds efficiently to PC/PE/PtdIns(4,5)P 2 and PC/PE/PtdIns(4,5)P 2 +YxxΦ cargo liposomes, resulting in GST-FCHO2 present in preformed complexes being excluded to the soluble fraction since it contains no membrane binding function. (C) Quantitation of three independent experiments in (B), showing that the ratio of FCHO2:AP2 is ~20-fold higher on beads than on PtdIns(4,5)P 2 -containing liposomes. ( D ) Tomographic structure of AP2 on the membrane in the presence of the FCHO2 linker. The cryo-EM map of AP2 recruited to cargo-free membranes in the presence of the FCHO2 linker, resolved to 9.7 Å, is shown as a gray isosurface. The map is fitted with the previously published ribbon model of AP2 bound to YxxΦ motif–containing membranes in the absence of the FCHO2 linker [Protein Data Bank (PDB) 6YAF]. Here and throughout, subunits are colored: α (dark blue), β2 (green), Nμ2 (dark purple), Cμ2 (pale purple), and σ2 (cyan). Molecular weight (MW).

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques: Binding Assay, Recombinant, Staining, Polyacrylamide Gel Electrophoresis, Liposomes, Membrane, Quantitation Assay, Cryo-EM Sample Prep, Molecular Weight

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( A ) Human FCHO2 (FC2 top) and FCHO1 (FC1 bottom) interdomain linker sequences aligned with identities highlighted in black and conservation in gray. Consensus CK2 phosphorylation sites are shown in dark pink and hierarchical CK2 sites in light pink. The conserved sequences blocks N1, N2, N3, and C assessed on the basis of conservations between sequences from across species and in the case of FCHO2, also on the basis of structure, are colored yellow, orange, red, and claret, respectively (coloring maintained throughout all subsequent figures). ( B ) Summary of the binding of the deletion constructs indicated on the unstructured FCHO2 and FCHO1 linkers to recombinant AP2 core and AP2 from brain cytosol as indicated. Gels shown in fig. S3.

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques: Phospho-proteomics, Binding Assay, Construct, Recombinant

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( A ) Structure of AP2:β2FCHO2linker chimera showing the position of the bound N3 helix (red) and InsP6 in orthogonal as indicated. ( B ) Left: Single-particle EM reconstruction of AP2:μ2FCHO2-N1+N2+N3 chimera highlighting the position of FCHO2-N1+N2 (see N1N2-enriched subclass in fig. S5 and table S6). The map, locally filtered with a global resolution of 7 Å, is colored according to AP2 subunit, the positions of which are derived from a fitted closed AP2 core (PDB 2vgl), and the approximate locations of N1 (yellow) and N2 (orange) are also indicated. N1 is persistent in all reconstructions of AP2:μ2FCHO2-N1+N2+N3 chimera, whereas N2 appears to be less fixed (fig. S5): N3 is not seen in any single-particle reconstructions. The position of N1 is in agreement with x-ray crystallographic structures . N2 appears to extend from the N1 density associated with the Cμ2 BR3 site across the broad, positively charged BR4 surface patch of Cμ2. Right: Ribbon representation of AP2 in the same orientation as the left panel with FCHO linker shown in worm representation.

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques: Single Particle, Derivative Assay

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( A ) AP2 core binding to GST, GST-FCHO2 WT, and phosphomimetic linker in pellet fraction (P). SDS-PAGE Coomassie-stained (top) and anti-μ2 subunit immunoblot (below). There is a clear loss of AP2core from the supernatant (S) fraction with either increased linker binding to phosphomimetic over FCHO2 WT linker in (P). ( B ) Affinity isolation of His 6 -MBP-Cμ2 by GST, GST-FCHO2 WT, and phosphomimetic linker in pellet fraction (P) shown by Coomassie staining of SDS-PAGE. The extent of Cμ2 binding in the pellet (P) fractions is enhanced by S→E phosphomimetic substitution. ( C ) Surface representations of the Cμ2 subdomain structure with positively charged residues from BR3 and BR4 highlighted in dark blue (left) and (right) same view of Cμ2 colored for electrostatic potential contoured from −0.5 V (red) to +0.5 V (blue). ( D ) WT or BR4-mutated (K339A, K341A, K343A, and K345A) His 6 -MBP-Cμ2 binding to GST, GST-SERINC3-L10, or GST-FCHO2 phosphomimetic linker as indicated by Coomassie blue staining of SDS-PAGE. Robust binding of SERINC3-L10 or GST-FCHO2 phosphomimetic linker is shown in (P), which is severely inhibited by the BR4- compound mutation. Note that the binding of GST-FCHO2 phosphomimetic linker is less affected than the binding of SERINC3-L10, confirming the presence of another binding site for the FCHO2 linker on Cμ2, i.e., BR3 patch. ( E ) Binding of GST-YxxΦ to AP2 core (blue) and AP2:μ2FCHO2-N1+N2+N3 chimera (red) in physiological buffer shown by BLI. AP2:β2FCHO2linker chimera shows ~3-fold tighter binding to TGN38 due to faster on and slower off rates than does AP2 core.

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques: Binding Assay, SDS Page, Staining, Western Blot, Isolation, Mutagenesis

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( A ) Overall structure of a single AP2 core in its open form bound to N1 and N2 from one FCHO linker and N3 from a different FCHO linker (N1 in yellow, N2 in orange, N3 in red, and TGN38 cargo in pink in all subsequent images). ( B ) Orthogonal views of three AP2s in crystals formed in the presence of FCHO. The N1 and N3 blocks of any given linker bind to different AP2 molecules. N1 binds to the N terminus of Cμ2, and N3 binds to the C terminus of β2 from a different AP2. ( C ) Close-up of the binding of N3 helix and preceding strand in hydrophobic troughs between helices 26 and 27 and between 22 and 24, respectively, of β2 in open AP2/FCHO2 linker complex crystals. ( D ) Close-up of the position of the binding site of N1 and N2 to Cμ2 in open AP2/FCHO2 linker complex crystals.

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques: Binding Assay

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( A ) Structure of α-appendage (blue) showing the FCHO1 linker C block (LFGPPLES) bound to the platform subdomain at 1.4-Å resolution. Key binding residues are indicated. The residues preceding the core FGPPL motif for a short helix that packs against the side of the platform subdomain. ( B and C ) Superposition of FCHO1 linker C block with (left) bound FxDxF peptide (PDB 1KYU7) and (right) bound DPF peptide (PDB 1KYU), both shown in semitransparent representation. The FCHO1 SKNLFGPPLES peptide binds in the opposite orientation to FxDxF and DPF peptides whose final F and central F, respectively, sit in the same hydrophobic pocket as the C-terminal L of LFGPPL. ( D ) ITC showing ~50 μM K D binding of LFGPPL to α-appendage. Mutation of FG to SS, LES to AAA, and PP to SS all reduces binding to weaker than 500 μM K D . ( E ) Top: 1.7-Å resolution structure of FCHO2 WxxL motif of the WDPLFGPSLDS C block (claret) bound to Cμ2 at its YxxΦ motif–binding site ( K D of 56 μM and electron density; see fig. S6) in perpendicular views. Bottom: Same perpendicular views of Cμ2 bound to TGN38 YxxΦ cargo peptide (pink).

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques: Blocking Assay, Binding Assay, Mutagenesis

Journal: Science Advances

Article Title: FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

doi: 10.1126/sciadv.abn2018

Figure Lengend Snippet: ( Top ) FCHO2 bound to the membrane uses its four linker blocks C, N3, N2, and N1 to engage with a closed, cytosolic AP2. Full binding results in the AP2 being orientated and concentrated on the membrane and destabilized to a “μ-out,” flexible conformation. This conformer quickly binds to the membrane, causing it to fully open and N1 and N2 to dissociate. Cargo binding stabilizes this open form, while the binding of polymerized clathrin and regulatory/accessory proteins competes out N3 and C, and the FCHO is expelled to the edge of the forming CCP where it can recruit further AP2s. This process occurs during the phases of CCP growth indicated. TDs, clathrin terminal domains. ( Middle ) Schematic representation perpendicular to the PM of the phases of a CCP’s life from initiation to scission. Left to right: FCHO2 in red; AP2 in blue; clathrin in green; other clathrin adaptors and accessory protein in gray. The pale blue gradient indicates an ~100-nm evanescent field as used for visualization in C. ( Bottom ) Schematic representation of projections of a CCP life cycle’s phases measured or inferred from eTIRF-SIM data. Top line, overlay of respective signals of FCHO2 (red), clathrin (green), and AP2 (blue).

Article Snippet: Genome editing of U-2 OS cells to generate isogenic knockout clones of endogenous FCHO2 (FCHO2-KO) was performed by Genscript.

Techniques: Membrane, Binding Assay