1. Volume 143, Issue 3, Pages 430-441 (October 2010)
Endophilin Functions as a Membrane-Bending Molecule and Is Delivered to Endocytic Zones by Exocytosis 
Jihong Bai, Zhitao Hu, Jeremy S. Dittman, Edward C.G. Pym, Joshua M. Kaplan 
Cell 
Volume 143, Issue 3, Pages (October 2010)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
The UNC-57 BAR Domain Promotes SV Endocytosis through Its Membrane Interactions
The phenotypes of wild-type (WT), unc-57(e406) endophilin mutants, and the indicated transgenic strains were compared. Transgenes were mCherry tagged UNC-57 variants, including full-length (FL; residues 1–379), BAR domain (residues 1–283), and ΔN (residues ). Transgenes were expressed in all neurons, using the snb-1 promoter. Expression levels of these transgenes are shown in Figure S1.
(A) Representative 1 min locomotion trajectories are shown (n = 20 animals for each genotype). The starting points for each trajectory were aligned for clarity. (B) Locomotion rates are compared for the indicated genotypes. Representative traces (C) and summary data for endogenous EPSC rates (D) are shown. Representative images (E) and summary data (F) for axonal SpH fluorescence in the dorsal nerve cord are shown for the indicated genotypes. The number of worms analyzed for each genotype is indicated. ∗∗, p < compared to WT controls. ##, p < when compared to unc-57 mutants. Error bars, standard error of the mean (SEM). See also Figure S1 and Figure S2.
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4. Figure 2
The Membrane-Bending Activity of Endophilin A BAR Domains Promotes SV Endocytosis
(A–C) Transgenes encoding wild-type and mutant BAR domains (1–247) from rEndoA1 BAR were analyzed for their ability to rescue locomotion rate (A), the surface Synaptobrevin (SpH) (Figure S3B), and EPSC rate (B and C) defects of unc-57 mutants. The ΔH1, A66W, and M70S,I71S mutations alter membrane tubulation activity but have little or no effect on membrane binding in vitro (Gallop et al., 2006; Masuda et al., 2006). All transgenes were tagged with mCherry at the C terminus to assess differences in expression levels (Figure S3).
(D) Transgenes expressing BAR domains derived from different proteins were compared for their ability to rescue the locomotion rate defect of unc-57 mutants. BAR domains are indicated as follows: rat endophilin A (rEndo A1, A2, and A3; residues 1-247); lamprey endophilin A (LampEndo; residues 1-248); C. elegans endophilin B (CeEndo B; residues 1-267); rat endophilin B (rEndo B; residues 1-247); and rat amphiphysin (ramphiphysin; residues 1-250).
(E) Alignment of the H1 helix sequence is shown for the indicated BAR domains. The A66 residue (green, arrow) is required for tubulation activity (Masuda et al., 2006). rEndo A3 has a sequence polymorphism (S64Y) compared to the A1 and A2 isoforms.
(F) Rescuing activities of rEndo A1, A2, A3, and A3(Y64S) BAR domains for the unc-57 mutant locomotion defect are compared. All transgenes were expressed in all neurons using the snb-1 promoter. The number of animals analyzed for each genotype is indicated. ∗∗ and ∗, significant differences compared to WT (p < and p < 0.01, respectively). ##, p < when compared to unc-57 mutants.
Error bars, SEM. See also Figure S3.
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5. Figure 3
Endophilin Is Targeted to the SV Pool at Presynaptic Terminals
Full-length unc-57 endophilin was tagged at the C terminus with mCherry and photoactivatable GFP (designated as CpG) (schematic shown in Figure 4A).
(A and B) The distribution of UNC-57::CpG mcherry fluorescence in DA neuron dorsal axons is compared with a coexpressed SV (GFP::SNB-1 [A]) or endocytic marker (APT-4::GFP AP2α [B]).
(C) Targeting of UNC-57::CpG to presynaptic terminals was strongly reduced in unc-104(e1265) KIF1A mutants.
See also Figure S4.
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6. Figure 4
Exocytosis Promotes Dissociation of Endophilin from the SV Pool
(A) Photoactivation of UNC-57::CpG at a single synapse is shown schematically (above) and in representative images (below).
(B and C) Representative images and traces of photoactivated UNC-57::CpG green fluorescence decay in wild-type (WT) and unc-13(s69) mutants. The mCherry fluorescence was captured to control for motion artifacts.
(D) Dispersion rates of photoactivated UNC-57::CpG were quantified in the indicated genotypes. Decay constants (τ) are 28.1 ± 3.3 s for WT; ± 13.6 s for unc-13 (s69); ± 26.4 s for unc-18 (e81); and 68.2 ± 6.5 s for tom-1(nu468)unc-13(s69).
Representative images (E) and summary data (F) for steady-state UNC-57::CpG mCherry fluorescence in the dorsal nerve cord axons were compared for the indicated genotypes. (F) Synaptic enrichment of UNC-57::CpG was calculated as follows: ΔF/F = (Fpeak − Faxon)/Faxon. The number of animals analyzed for each genotype is indicated. ∗∗, p < compared to WT controls.
Error bars, SEM. See also Figure S5.
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7. Figure 5 Structural Requirements for UNC-57 Regulation by Exocytosis
Representative traces and summary data are shown comparing the dispersion of mutant UNC-57 proteins. Mutant proteins analyzed are: (A) WT BAR domain lacking the SH3 (BAR reporter), and full-length UNC-57 proteins containing the ΔN (membrane-binding deficient) (B); A66W (tubulation deficient) (C); and ΔH1I (dimerization deficient) (D) mutations. Each mutant protein was tagged with CpG, expressed in DA neurons, and their dispersion rates compared following photoactivation in wild-type and unc-13 mutants. ∗∗, p < compared to WT controls. n.s., nonsignificant. Error bars, SEM. See also Figure S6.
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8. Figure 6
RAB-3 and the Rab3 GEF (AEX-3) Regulate Endophilin Targeting to SVs
Representative images (A) and quantification (B) of UNC-57::CpG synaptic enrichment in WT, aex-3, unc-13, and unc-13;aex-3 double mutants are shown (Synaptic enrichment: WT 8.6 ± 0.6; aex ± 0.6; unc-13; aex ± 1.2, unc ± 2.2-fold). Dispersion rates of UNC-57::CpG in WT (τ = 28.1 ± 3.3 s) and aex-3 mutant (τ = 45.5 ± 5.1 s) animals were compared in (C). (D and E) UNC-57::CpG distribution in transgenic unc-13 mutant animals with overexpressed RAB-3 (Q81L) or (T36N) was studied. Overexpression of RAB-3 (Q81L), but not RAB-3 (T36N), significantly reduced UNC-57::CpG synaptic enrichment in unc-13 mutants. ∗∗, p < and ∗, p < 0.01, compared to WT controls. Error bars, SEM.
Cell  , DOI: ( /j.cell )
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9. Figure 7 Analysis of a Membrane-Anchored UNC-57 Protein
(A) The distribution of UNC-57(PM) in DA neuron axons was compared with coexpressed UNC-57::CpG (upper panels), active zone [AZ] marker ELKS-1::mcherry (middle panels), or endocytic zone [EZ] marker APT-4::mcherry (AP2α, lower panels). UNC-57(PM) comprises full-length UNC-57 and GFP fused to the N-terminus of UNC-64 Syntaxin 1A (schematic shown in Figure S7A).
(B) GFP fluorescence of UNC-57(PM) in WT and unc-13(s69) mutant animals were quantified. UNC-57(PM) was expressed in all neurons with the snb-1 promoter.
(C) Representative images are shown of wild-type and mutant UNC-57(PM) proteins in dorsal cord axons. The BAR(PM) protein corresponds to UNC-57(PM) lacking the SH3 domain. The ΔN(PM) protein lacks the N-terminal 26 residues of UNC-57, which prevents membrane binding.
(D) Representative traces of endogenous EPSC from WT, unc-57(e406) mutants, and transgenic unc-57 animals carrying wild-type and mutant UNC-57(PM) constructs.
Endogenous EPSC rates (left panel) and amplitudes (right panel) are shown in (E). Significant differences (p < by Student's t test) are indicated as: ∗∗, compared to WT; and ##, compared to unc-57 mutants. n.s., nonsignificant.
Error bars, SEM. See also Figure S7.
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10. Figure S1
Properties of Transgenic Animals Carrying UNC-57 Constructs, Related to Figure 1
(A) Summary data for endogenous EPSC amplitudes are shown for the indicated genotypes. No significant differences were observed.
(B) Expression levels of WT and mutant UNC-57 constructs are analyzed. Representative images of mcherry fluorescence in the dorsal cord axons (above) and integrated axonal fluorescence intensities (in a 10 μm window) are shown (below). BAR and ΔN constructs were expressed at 1.07 ± 0.05 and 1.25 ± 0.07 fold of the full length UNC-57 level, respectively.
(C) Quantitation of unc-57 mRNA levels is shown. A schematic drawing of the unc-57 gene structure is shown (above). Two primer pairs were designed to target adjacent exons (indicated as black arrows). qPCR was carried out as described in the Methods. Levels of unc-57 mRNA in unc-57(e406) and transgenic animals carrying Psnb-1::unc-57 BAR::mcherry (nuIs322;unc-57) were normalized to wild-type levels and plotted in lower panel. Both primer sets reported reduced mRNA levels in unc-57 mutants (0.39 and 0.48 fold, respectively; compared with WT), presumably due to non-sense mediated mRNA decay. In nuIs322;unc-57 animals, mRNA levels were increased (2.39 and 2.55 fold, respectively), suggesting that the nuIs322 transgene leads to a 2-fold increase in unc-57 mRNA level. All constructs in this figure contain the snb-1 promoter.
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11. Figure S2
Endophilin-Scaffolding Functions Are Not Required for SV Endocytosis, Related to Figure 1
Representative images (A) and summary data (B) for GFP::UNC-26 puncta fluorescence in the dorsal cord are shown for the indicated genotypes. (C) An UNC-26 synaptojanin transgene lacking the PRD (deletion of residues ) rescues the locomotion rate defect of unc-26(s1710) mutants. FL indicates full-length UNC-26. (D and E) GFP::synaptojanin Fluorescence Recovery After Photobleaching (FRAP). GFP::UNC-26 synaptojanin fluorescence in a 10 μm axonal region was photobleached with a 488nm laser. Recovery of GFP fluorescence was followed for 50 s. Representative images (D) and recovery trace (E) are shown. Immobile fractions of GFP::UNC-26 synaptojanin are 49 ± 2%, 53 ± 6% and 49 ± 2% in WT, unc-57 mutants and rescued animals expressing UNC-57 BAR, respectively (F). Representative images and summary data for GFP tagged DYN-1 Dynamin (DYN-1::GFP) (G and H) and summary data for AP2α-subunit (APT-4::GFP) (I) are shown. Quantified puncta fluorescence intensities (H and I) were normalized to levels observed in wild-type controls. “n.s.” indicates statistically non-significant. (∗∗) and (∗) indicate significant differences compared to WT (p < and p < 0.01, respectively). (##) indicates p < when compared to unc-57 mutants. Error bars indicate SEM.
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12. Figure S3
Analysis of Endophilin Tubulation Mutants, Related to Figure 2
(A) Expression levels of WT and mutant versions of rat endophilin (rEndo) BAR domains were quantified using mcherry fluorescence (as in Figure S1).
(B and C) Transgenes encoding wild-type and mutant rEndo BAR domains were analyzed for their ability to rescue defects in surface Synaptobrevin (SpH) (B) and endogenous EPSC amplitudes (C).
(D) Cumulative distributions of endogenous EPSC amplitudes are shown for WT (black) and rEndo BAR A66W (red).
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13. Figure S4
Comparison of UNC-57 and RAB-3 Mobilities, Related to Figure 3
(A) The distribution of UNC-57 endophilin::CpG (Red) in DA motor neuron axons was compared with a coexpressed SV marker (GFP::RAB-3).
(B) Photoactivated UNC-57::CpG was recaptured at adjacent synapses. UNC-57::CpG in synapse #1 was photoactivated. Fluorescence changes in synapse #1, two adjacent synapses (#2 and #3) and axonal region (indicated as A) were followed and plotted.
(C) Time-courses of GFP fluorescence recovery of either UNC-57::GFP or GFP::RAB-3 (within 2 μm window) after photo-bleach are shown.
(D) Summary data of recovery fractions (after 25 s) are plotted for UNC-57 and RAB-3.
Error bars indicate SEM.
Cell  , DOI: ( /j.cell )
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14. Figure S5
Effects of Exocytosis on UNC-57::CpG Distribution and Dynamics Are Cell Autonomous, Related to Figure 4
(A and B) Expression of Tall Syntaxin (tall syx) significantly decreased the UNC-57::CpG dispersion rate (tall syx 49.1 ± 3.3 s, WT 28.1 ± 3.3 s; p < 0.001) (A) and significantly increased the synaptic enrichment of UNC-57::CpG (tall syx 16.4 ± 1.0, WT 8.6 ± 0.6; p < 0.001) (B). In panel B, representative images (above), cumulative distributions of synaptic enrichment (ΔF/F) for wild-type (black) and transgenic animals expressing Tall Syntaxin (red) (below left), and mean values of synaptic enrichment (below right) are shown. ∗∗ indicates p < 0.001, compared to WT controls. Error bars indicate SEM.
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15. Figure S6
UNC-57 Mutants Remain Enriched at Synapses, Related to Figure 5
Representative images of mutant UNC-57::CpG constructs in dorsal axons of DA neurons are shown in (A). Synaptic enrichment of these constructs are quantified in (B). Synaptic enrichment of UNC-57::CpG FL, BAR, ΔN and ΔH1I constructs are 8.6 ± 0.6 fold, 8.9 ± 0.8 fold, 4.6 ± 0.4 fold and 5.9 ± 0.3 fold, respectively. ∗∗ indicates p < 0.001, compared to full length UNC-57::CpG (FL) controls. Error bars indicate SEM.
Cell  , DOI: ( /j.cell )
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16. Figure S7
Endophilin Remains Fully Active When Tethered to the Cytoplasmic Domain of Syntaxin1A, Related to Figure 7
Schematic drawings of constructs that constitutively target WT and mutant UNC-57 to the plasma membrane are shown in (A). GFP is used to visualize localization of these constructs. For plasma membrane targeting, full length UNC-64 Syntaxin1A was tagged to the C terminus of these constructs. As a control, a truncated UNC-64 that lacks the transmembrane region (residues ) was also tagged to the C terminus of UNC-57::GFP (designated as UNC-57(cyto).
(B) GFP::UNC-64 exhibits diffused pattern on axonal plasma membrane of the DA neurons.
(C) Representative images and summary data of UNC-57(cyto) in WT and unc-13 animals. In contrast to UNC-57(PM) shown in Figure 7, synaptic enrichment of UNC-57(cyto) increases in unc-13 animals.
(D) Locomotion rates are compared for the indicated genotypes.
(E and F) Electrophysiological recordings from transgenic unc-57 animals that express UNC-57(cyto). Expression of UNC-57(cyto) fully rescued defects in endogenous EPSC rates and had no effect on endogenous EPSC amplitudes. Significant differences (p < by Student's t test) are indicated as: ∗∗, compared to WT. Error bars indicate the SEM.
Cell  , DOI: ( /j.cell )
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