Fig 1. Fwe controls Clathrin-Mediated Endocytosis (CME) in a channel-independent manner.

(A–G) Confocal Z-projection images of neuromuscular junction (NMJ) boutons labeled with fixable FM1-43 dye were obtained from FRT80B control larvae (A), fwe mutant larvae (fwe^DB25/fwe^DB56, B), 50% Fwe-rescued larvae (nSyb > flag-fwe-HA in fwe^DB25/fwe^DB56, C), 50% FweE79Q-rescued larvae (nSyb > flag-fweE79Q-HA in fwe^DB25/fwe^DB56, D), 10% Fwe-rescued larvae (elav > flag-fwe-HA in fwe^DB25/fwe^DB56, E), 4% Fwe-rescued larvae (nSyb(w) > flag-fwe-HA in fwe^DB25/fwe^DB56, F) and 4% FweE79Q-rescued larvae (nSyb(w) > flag-fweE79Q-HA in fwe^DB25/fwe^DB56, G). FM1-43 dye uptake was elicited by 1-min 90 mM K⁺/0.5 mM Ca²⁺ stimulation. (H) The absolute dye fluorescence intensities in the boutons were measured and normalized to the average value of controls. Loss of fwe significantly impairs dye uptake, which can be restored by expressing different levels of wild-type Fwe or FweE79Q protein. Type Ib boutons derived from A2 muscles 6/7 were counted, and NMJs (control, n = 8; fwe mutant, n = 6; 50% Fwe, n = 6; 50% FweE79Q, n = 6; 10% Fwe, n = 6; 4% Fwe, n = 8; and 4% FweE79Q, n = 6) derived from 5 larvae for each genotype were analyzed. (I–P) Transmission electron microscopy (TEM) images of NMJ boutons were obtained from larvae of the indicated genotypes. They were processed under the resting condition (10-min incubation in 5 mM K⁺/0 mM Ca²⁺ solution, I–L) or after 10-min 60 mM K⁺/1 mM Ca²⁺ stimulation (M–P). White arrows indicate individual synaptic vesicles (SVs). (Q) Data quantifications of SV density. A low SV number was found in fwe mutants, which is corrected in the presence of 4% Fwe and 4% FweE79Q. Ten-minute 60 mM K⁺/1 mM Ca²⁺ stimulation further lowers the SV density in fwe mutants. Type Ib boutons (at rest: control, n = 14; fwe mutant, n = 23; 4% Fwe, n = 19; and 4% FweE79Q, n = 13. Ten-minute 60 mM K⁺/1 mM Ca²⁺ stimulation: control, n = 13; fwe mutant, n = 36; 4% Fwe, n = 14; and 4% FweE79Q, n = 24) derived from 3 larvae for each genotype were analyzed. (R) Data quantifications of SV size. Enlarged SV size was found in fwe mutants, which is corrected in the presence of 10% Fwe, 4% Fwe, or 4% FweE79Q. SVs (control, n = 1,675; fwe mutants, n = 780; 10% Fwe, n = 2,196; 4% Fwe, n = 1,243; and 4% FweE79Q, n = 857) obtained from ≥10 type Ib boutons were analyzed. Boutons were derived from ≥3 larvae for each genotype. All statistical analyses used a one-way ANOVA test, except the comparison of the SV number at rest and after stimulation, which was performed using a Student’s t test. p-Value: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.01; ****, p < 0.001. Error bars indicate the standard error of the mean. Scale bar: 5 μm in A–G; 500 nm in I–P. The underlying data can be found in S1 Data.