Fig 6. Rodent Fwe isoform 2 is localized on the Synaptic Vesicles (SVs) and triggers Clathrin-Mediated Endocytosis (CME) and Activity-Dependent Bulk Endocytosis (ADBE) at the Drosophila Neuromuscular Junction (NMJ).

(A) In semiquantitative reverse-transcription PCR (RT-PCR) assays, the expression of ratFwe2 mRNA was detected in distinct regions of adult rat brain. The PCR products of ratFwe2 cDNA were sequenced to validate the identity of ratFwe2 mRNA. The expression of GAPDH mRNA was used as the internal control. (B) In immunoblots with α-m/ratFwe2, mFwe2 was detected in the lysates of mouse neuroblastoma Neuro 2a (n2a) cells. This blotting signal is further reduced by expressing either one of two different mFwe-microRNAi (miRNAi). This isoform is also present in postnatal day 16 and adult mouse brains. Actin was used as the loading control. (C) In the immunoblots with α-m/ratFwe2, ratFwe2 was detected in the embryonic rat brain as well as in different days in vitro (DIV) cultured rat hippocampal neurons. Actin was used as the loading control. (D) The subcellular fractions were obtained from adult rat brain extracts using a series of centrifugations. ratFwe2 was found in the synaptosomal plasma membrane (lysate pellet 1 [LP1]) and SV fractions (lysate pellet 2 [LP2]) but was not detected in the cytosolic fraction (lysate supernatant 2 [LS2]). * indicates a degraded product of ratFwe2 or nonspecific antibody binding. (E) The subcellular fractions of adult rat brain were separated by 0.3–0.99 M sucrose gradients. ratFwe2 is present in the SV fractions containing Synaptophysin (Syp). Immunoblotting for GM130, a Golgi protein, labels Golgi fractions. (F–G) Confocal Z-projection images of DIV14 cultured rat hippocampal neurons stained with α-HA (blue), α-Syp (red), and α-GFP (green) were captured from neurons transfected with pSpCas9(BB)-m/ratFwe-gRNA-2A-GFP-2A-mFwe2-HA plasmid. White arrows indicate the presynaptic terminals. The enlarged images for white dashed boxes in F are shown in G. Axons are outlined by α-GFP staining. mFwe2-HA proteins are largely colocalized with Syp in the presynaptic terminals. (H–H2) Confocal Z-projection images of Drosophila NMJ boutons stained with α-HRP (red) and α-HA (green) were obtained from fwe mutants expressing Flag-mFwe2-HA (nSyb(w) > flag-mFwe2-HA in fwe^DB25/fwe^DB56). (I–J) Confocal Z-projection images of Drosophila NMJ boutons labeled with fixable FM1-43 dye were obtained from FRT80B control larvae (I) and Flag-mFwe2-HA-rescued larvae (J). CME was elicited by 1-min 90 mM K⁺/0.5 mM Ca²⁺ stimulation in the presence of 4 μM fixable FM1-43 dye. (K) Data quantifications for the FM1-43 dye intensity within the type Ib boutons shown in I–J. The FM1-43 dye fluorescence intensities were measured, and the values are normalized to the average value of controls. The neuronal expression of Flag-mFwe2-HA rescues the FM1-43 dye uptake defect in fwe mutants. Type Ib boutons of A2 muscles 6/7 were counted, and NMJs (control, n = 7; and Flag-mFwe2-HA rescue, n = 8) derived from 4 larvae were analyzed. (L–N) Transmission electron microscopy (TEM) images of NMJ boutons were captured from Flag-mFwe2-HA-rescued larvae. Samples were fixed under resting condition (10-min incubation in 5 mM K⁺/0 mM Ca²⁺ solution, L) or after 10-min 90 mM K⁺/2 mM Ca²⁺ stimulation (M). Data quantifications of the number of bulk cisternae per bouton area are shown in N. Type Ib boutons (at rest, n = 10; and 10-min 90 mM K⁺/2 mM Ca²⁺, n = 20) from 3 larvae for each condition were analyzed. A Student’s t test was used for statistical analysis. p-Value: ns, not significant; ****, p < 0.001. Error bars indicate the standard error of the mean. Scale bar: 2 μm (G–G3); 5 μm (F–F3, H–H2); 10 μm (I–J); 500 nm (L–M). The underlying data can be found in S1 Data.