Neuroscience

E3 Ubiquitin Ligase Highwire/Phr1 Phase Separation Mediates Endocytic Control of JNK Signaling in Drosophila Neurons

Srikanth Pippadpally author has email address
Anjali Bisht
Saumitra Dey Choudhury
Manish Kumar Dwivedi
Zeeshan Mushtaq
Suneel Reddy-Alla
Vimlesh Kumar author has email address

Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, India
College of Community Science, Acharya N.G. Ranga Agricultural University, Guntur, India

https://doi.org/10.7554/eLife.109510.1

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Figures and data

Compromised endocytosis causes Hiw to accumulate in large assemblies within the neuronal cell body
(A) Schematic representation of the third instar larval ventral nerve cord (VNC). The magnified view represents accumulation and possible mislocalization of the Hiw assemblies in the neuronal cell bodies of endocytic mutants. (B-G’’) Confocal images of the neuronal cell body in third instar larval VNC co-labeled for GFP-Hiw (green) and Elav (magenta) in GFP control (Elav-Gal4>UAS-GFP) (B-B’’), Elav-Gal4>UAS-GFP-Hiw (C-C’’), Elav-Gal4>UAS-GFP; AP2σ-^KG02457/AP2σ^ang7 (D-D’’), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 (E-E’’), Elav-Gal4>UAS-GFP-Hiw; UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (F-F’’), and Elav-Gal4>UAS-GFP-Hiw; synj1^1/2(G-G’’). The scale bar in G’’ represents 5 µm. (H) Histogram showing the average intensity of GFP-Hiw in third instar larval VNC in GFP control (Elav-Gal4>UAS-GFP) (1.07 ± 0.03), Elav-Gal4>UAS-GFP-Hiw (0.95 ± 0.08), Elav-Gal4>UAS-GFP; AP2σ-^KG02457/AP2σ^ang7(1.10 ± 0.04), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 (2.52 ± 0.10), Elav-Gal4>UAS-GFP-Hiw; UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (0.97 ± 0.07), and Elav-Gal4>UAS-GFP-Hiw; synj1^1/2 (1.94 ± 0.13). At least 10 VNCs of each genotype were used for the quantifications. (I) Histogram showing the average intensity of GFP-Hiw in individual neuronal cell body in GFP control (Elav-Gal4>UAS-GFP) (1.65 ± 0.08), Elav-Gal4>UAS-GFP-Hiw (0.45 ± 0.02), Elav-Gal4>UAS-GFP; AP2σ-^KG02457/AP2σ^ang7 (1.53 ± 0.11), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 (1.13 ± 0.04), Elav-Gal4>UAS-GFP-Hiw; UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (0.62 ± 0.02), and Elav-Gal4>UAS-GFP-Hiw; synj1^1/2 (1.15 ± 0.09). At least 10 VNCs of each genotype were used for the quantifications. (J) Histogram showing the number of GFP-Hiw puncta in GFP control (Elav-Gal4>UAS-GFP) (7.00 ± 1.48), Elav-Gal4>UAS-GFP-Hiw (806.6 ± 35.31), Elav-Gal4>UAS-GFP; AP2σ-^KG02457/AP2σ^ang7 (8.500 ± 1.37), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 (159.3 ± 16.52), Elav-Gal4>UAS-GFP-Hiw; UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (851.9 ± 28.73), and Elav-Gal4>UAS-GFP-Hiw; synj1^1/2 (150.9 ± 7.66). At least 10 VNCs of each genotype were used for the quantifications. (K) Histogram showing the size of GFP-Hiw puncta in GFP control (Elav-Gal4>UAS-GFP) (0.01 ± 0.002), Elav-Gal4>UAS-GFP-Hiw (0.06 ± 0.01), Elav-Gal4>UAS-GFP; AP2σ-^KG02457/AP2σ^ang7 (0.01 ± 0.001), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 (0.20 ± 0.01), Elav-Gal4>UAS-GFP-Hiw; UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (0.11 ± 0.01), and Elav-Gal4>UAS-GFP-Hiw; synj1^1/2(0.21 ± 0.004). At least 10 VNCs of each genotype were used for the quantifications. The error bar in H, I, J, and K represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001, ns: not significant. (L) Western blot showing the level of GFP-Hiw in control (Elav-Gal4>UAS-GFP-Hiw), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7, Elav-Gal4>UAS-GFP-Hiw; UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7, and Elav-Gal4>UAS-GFP-Hiw; synj1^1/2. Ran was used as a loading control. (M) Histogram showing the level of GFP-Hiw in control (Elav-Gal4>UAS-GFP-Hiw) (1.0 ± 0.00), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7(1.73 ± 0.06), Elav-Gal4>UAS-GFP-Hiw; UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (1.02 ± 0.12), and Elav-Gal4>UAS-GFP-Hiw; synj1^1/2 (1.53 ± 0.02). Three independent Western blots were used for quantification. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001; **p<0.01.

Highwire assembles as dynamic condensates in endocytic mutants
(A-C) The PLAAC scores of GFP, Atx2, and Hiw. The PLAAC scores represent the ability of a protein segment to form aggregates or LLPS (LANCASTER et al. 2014). The plots represent predicted intrinsically disordered regions in a protein. The red line denotes the likelihood of a protein segment being disordered. The blue line denotes the probability of a given position in a protein being a part of a disordered binding region. (D) The domain organization of Hiw protein. It contains an N-terminal RCC1-like domain (RLD), two (PAM-Hiw-RPM-1) PHR domains, followed by (Rae1-binding domain) RBD and (Fsn-1 binding domain) FBD1, and a C-terminal (RING H2 ubiquitin-ligase domain) RING domain. (E-L’’) Confocal images of the neuronal cell body co-labelled for GFP-Hiw (green) and Elav (magenta) in control (Elav-Gal4>UAS-GFP-Hiw) and Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 treated with 0% 1,6-HD (E-F’’), 1% 1,6-HD (G-H’’), 5% 1,6-HD (I-J’’), and 10% 1,6-HD (K-L’’) containing Schneider’s media for 2 mins. The 0% contains only media. The scale bar in L’’ represents 5 µm for images E-L’’. (M-O’’) Confocal images of the neuronal cell body co-labelled for GFP-Hiw (green) and Elav (magenta) in Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 treated with 1% 2,5-HD (M-M’’), 5% 2,5-HD (N-N’’), and 10% 2,5-HD (O-O’’) containing Schneider’s media for 2 mins. The scale bar in O’’ represents 5 µm for images M-O’’. (P) Line graph showing the average size of the GFP-Hiw punctae in control (Elav-Gal4>UAS-GFP-Hiw) (black line) and Elav-Gal4>UAS-GFP-Hiw; AP2σ^KG02457/AP2σ^ang7 treated with 0%, 1%, 5% and 10% 1,6-HD (blue line), and Elav-Gal4>UAS-GFP-Hiw; AP2σ^KG02457/AP2σ^ang7 treated with 1%, 5% and 10% 2,5-HD (orange line). (Q-R’’) Confocal images of the neuronal cell body co-labelled for GFP-Hiw (green) and Elav (magenta) in Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 treated with 5% 1,6-HD for 2 min and allowed to recover for 2 hrs after removal of 1,6-HD in S2 Schneider’s media. The scale bar in R’’ represents 5 µm. (S) Histogram showing the size (in µm) of GFP-Hiw puncta in Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 treated with 5% 1,6-HD for 2 min (0.076 ± 0.005), and Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 after recovery for 2 hrs (0.133 ± 0.003). At least 8 VNCs of each genotype were used for the quantifications. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using Student’s t-test. ***p<0.001.

Acute inhibition of endocytosis triggers Hiw/Phr1 condensate formation
(A-I’) Confocal images of the neuronal cell body co-labelled for GFP-Hiw (green) and Elav (magenta) in control (Elav-Gal4>UAS-GFP-Hiw) (A-C’), Elav-Gal4>UAS-GFP-Hiw; shi^ts1 (D-F’), and Elav-Gal4>UAS-GFP-Hiw; shi^ts2 (G-I’). The scale bar in I’ represents 5 µm. (J) Histogram showing the average size of GFP-Hiw puncta in control, Elav-Gal4>UAS-GFP-Hiw; shi^ts1, and Elav-Gal4>UAS-GFP-Hiw; shi^ts2 at permissive (20 °C) and restrictive temperatures (29 °C). At least 8 VNCs of each genotype were used for the quantifications. (K-P’) Confocal images of the neuronal cell body co-labelled for GFP-Hiw (green) and Elav (magenta) in Elav-Gal4>UAS-GFP-Hiw, and Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 treated with 5% 2,5-HD and 1,6-HD for 2 min. The scale bar in P’ represents 5 µm. (Q) Histogram showing the average size of GFP-Hiw puncta in untreated, 5% 2,5-HD, and 5% 1,6-HD treated control, Elav-Gal4>UAS-GFP-Hiw; shi^ts1. At least 8 VNCs of each genotype were used for the quantifications.

Endocytosis restricts synaptic growth via MAP3K/Wnd signaling
(A-F’’) Confocal images of third instar larval ventral nerve cord co-labelled for Wnd (magenta) and NC82 (green) in control (A-A’’), Elav-Gal4>UAS-GFP-Hiw (B-B’’), AP2σ-^KG02457/AP2σ^ang7(C-C’’), Elav-Gal4>UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7(D-D’’), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 (E-E’’), and (F-F’’) Elav-Gal4>UAS-GFP-Hiw; synj1^1/2. The scale bar in F’’ represents 20 µm. (G) Histogram showing the average fluorescence intensity of Wnd in control (0.76 ± 0.04), Elav-Gal4>UAS-GFP-Hiw (0.51 ± 0.01), AP2σ-^KG02457/AP2σ^ang7(1.46 ± 0.05), Elav-Gal4>UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (0.70 ± 0.05), Elav-Gal4>UAS-GFP-Hiw; AP2σ-^KG02457/AP2σ^ang7 (1.42 ± 0.04), and Elav-Gal4>UAS-GFP-Hiw; synj1^1/2 (1.14 ± 0.04). At least 8 VNCs of each genotype were used for the quantification. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. **p<0.01, ***p<0.001; ns, not significant. (H-N’’) Confocal images of muscle 6/7 NMJ at A2 hemisegment showing synaptic growth in control (H-H’’), wnd^2/3 (I-I’’), AP2σ-^KG02457/AP2σ^ang7 (J-J’’), Elav-Gal4>UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7(K-K’’), synj1^1/2 (L-L’’), wnd^2/3, AP2σ-^KG02457/AP2σ^ang7(M-M’’), and synj1^1/2; wnd^2/3 (N-N’’) immunolabeled for CSP (magenta) and HRP (green). The scale bar in N’’ represents 10 µm. (O) Histogram showing average bouton number from muscle 6/7 NMJ at A2 hemisegment for control (1.53 ± 0.07), wnd^2/3 (0.88 ± 0.04), AP2σ-^KG02457/AP2σ^ang7 (2.31 ± 0.13), Elav-Gal4>UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (1.68 ± 0.14), synj1^1/2 (2.46 ± 0.20), wnd^2/3, AP2σ-^KG02457/AP2σ^ang7(1.76 ± 0.13) and synj1^1/2; wnd^2/3 (2.15 ± 0.21). At least 8 NMJs of each genotype were used for the quantifications. (P) Histogram showing the average bouton area from muscle 6/7 NMJ at A2 hemisegment for control (6.56 ± 0.39), wnd^2/3 (10.6 ± 0.59), AP2σ-^KG02457/AP2σ^ang7 (1.16 ± 0.17), Elav-Gal4>UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (5.83 ± 0.23), synj1^1/2 (1.31 ± 0.12), wnd^2/3, AP2σ-^KG02457/AP2σ^ang7(4.39 ± 0.47) and synj1^1/2; wnd^2/3 (3.73 ± 0.28). At least 8 NMJs of each genotype were used for the quantifications. The error bar in O and P represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001; **p<0.01; *p<0.05.

Endocytosis restricts hyperactivation of JNK signaling
(A) Schematic illustration of the JNK signaling pathway in Drosophila. Autophagy selectively degrades Hiw, leading to elevated levels of MAP3K Wallenda, eventually resulting in Fos and Jun (AP-1) phosphorylation. Phosphorylation of AP-1 results in activation of JNK signaling. TRE-DsRed (TRE is a synthetic promoter containing AP-1 binding sites) and puc-LacZ (puckered is a target gene of JNK signaling) were used as reporters to analyze the activation of the JNK signaling pathway. (B-E’’) Confocal images of third instar larval ventral nerve cord co-labelled for β-Gal (magenta) and Elav (green) in control (puc-LacZ/+) (B-B’’), puc-LacZ, AP2σ-^KG02457/AP2σ^ang7(C-C’’), Elav-Gal4>UAS-AP2σ; puc-LacZ; AP2σ-^KG02457/AP2σ^ang7, (D-D’’) and synj1^1/2; puc-LacZ/+ (E-E’’). The scale bar in E’’ represents 20 µm. (F) Histogram showing the β-Gal levels in the third instar larval ventral nerve cord in control (puc-LacZ/+) (0.59 ± 0.02), puc-LacZ, AP2σ-^KG02457/AP2σ^ang7 (1.24 ± 0.06), Elav-Gal4>UAS-AP2σ; puc-LacZ; AP2σ-^KG02457/AP2σ^ang7 (0.48 ± 0.02), and synj1^1/2; puc-LacZ/+ (1.43 ± 0.07). At least 9 VNCs of each genotype were used for the quantification. (G) Histogram showing the β-Gal levels in the individual neuronal nuclei in control (puc-LacZ/+) (0.99 ± 0.09), puc-LacZ, AP2σ-^KG02457/AP2σ^ang7 (5.92 ± 0.37), Elav-Gal4>UAS-AP2σ; puc-LacZ; AP2σ-^KG02457/AP2σ^ang7 (1.14 ± 0.16), and synj1^1/2; puc-LacZ/+ (5.69 ± 0.36). At least 27 nuclei of each genotype were used for the quantification. The error bar in G and H represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001; ns: not significant. (H-K’’) Confocal images of third instar larval ventral nerve cord co-labelled for TRE-DsRed (magenta) and Elav (green) in control (TRE-DsRed/+), TRE-DsRed/+; AP2σ-^KG02457/AP2σ^ang7 (H-H’’), Elav-Gal4> UAS-AP2σ; TRE-DsRed/+; AP2σ-^KG02457/AP2σ^ang7 (I-I’’), and TRE-DsRed/+, synj1^1/2 (K-K’’). The scale bar in K’’ represents 20 µm. (L) Histogram showing the TRE-DsRed levels in the third instar larval ventral nerve cord in control (TRE-DsRed/+) (1.09 ± 0.08), TRE-DsRed/+; AP2σ-^KG02457/AP2σ^ang7(2.17 ± 0.18), Elav-Gal4> UAS-AP2σ; TRE-DsRed/+; AP2σ-^KG02457/AP2σ^ang7 (0.98 ± 0.08), and TRE-DsRed/+, synj1^1/2 (1.80 ± 0.21). At least 9 VNCs of each genotype were used for the quantification. (M) Histogram showing the TRE-DsRed levels in individual neuronal nuclei in control (TRE-DsRed/+) (2.19 ± 0.18), TRE-DsRed/+; AP2σ-^KG02457/AP2σ^ang7(3.56 ± 0.43), Elav-Gal4> UAS-AP2σ; TRE-DsRed/+; AP2σ-^KG02457/AP2σ^ang7 (1.18 ± 0.13), and TRE-DsRed, synj1^1/2(3.34 ± 0.26). At least 47 nuclei of each genotype were used for the quantification. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001; *p<0.05; ns, not significant.

Elevated BMP and JNK signaling together promote synaptic overgrowth in endocytic mutants
(A-H’’) Confocal images of muscle 6/7 NMJ at A2 hemisegment showing synaptic growth in control (A-A’’), AP2σ-^KG02457/AP2σ^ang7 (B-B’’), Elav-Gal4>UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7(C-C’’), Elav>UAS-kay^DN (D-D’’), Elav-Gal4>UAS-bsk^DN; AP2σ-^KG02457/AP2σ^ang7 (E-E’’), Elav-Gal4>UAS-kay^DN; AP2σ-^KG02457/AP2σ^ang7 (F-F’’), tkv⁷/+; AP2σ-^KG02457/AP2σ^ang7 (G-G’’), Elav-Gal4>UAS-kay^DN,tkv⁷; AP2σ-^KG02457/AP2σ^ang7 (H-H’’) immunolabeled for synaptic vesicle marker CSP (magenta) and neuronal membrane marker HRP (green). The scale bar in H’’ represents 10 µm. (I) Histogram showing average bouton number from muscle 6/7 NMJ at A2 hemisegment in in control (w¹¹¹⁸) (1.25 ± 0.10), AP2σ-^KG02457/AP2σ^ang7 (4.0 ± 0.37), Elav-Gal4>UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (1.61 ± 0.34), Elav>UAS-kay^DN (0.98 ± 0.06), Elav-Gal4>UAS-bsk^DN; AP2σ-^KG02457/AP2σ^ang7 (2.62 ± 1.83), Elav-Gal4>UAS-kay^DN; AP2σ-^KG02457/AP2σ^ang7 (2.21 ± 0.21), tkv⁷/+; AP2σ-^KG02457/AP2σ^ang7 (2.26 ± 0.10), Elav-Gal4>UAS-kay^DN,tkv⁷; AP2σ-^KG02457/AP2σ^ang7(1.88 ± 0.25). At least 9 NMJs of each genotype were used for the quantifications. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001; **p<0.01; ns, not significant. (J) Histogram showing the average bouton area from muscle 6/7 NMJ at A2 hemisegment for control (8.75 ± 0.45), AP2σ-^KG02457/AP2σ^ang7 (1.50 ± 0.18), Elav-Gal4>UAS-AP2σ, AP2σ-^KG02457/AP2σ^ang7 (7.81 ± 0.42), Elav>UAS-kay^DN (7.70 ± 0.28), Elav-Gal4>UAS-bsk^DN; AP2σ-^KG02457/AP2σ^ang7 (6.80 ± 0.50), Elav-Gal4>UAS-kay^DN; AP2σ-^KG02457/AP2σ^ang7 (7.78 ± 0.65), tkv⁷/+; AP2σ-^KG02457/AP2σ^ang7(5.48 ± 0.42), Elav-Gal4>UAS-kay^DN,tkv⁷; AP2σ-^KG02457/AP2σ^ang7(8.94 ± 0.42). At least 10 NMJs of each genotype were used for the quantifications. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001; *p<0.05; ns, not significant.

Loss of Rab11 induces Highwire accumulation and liquid-liquid phase-separation in neuronal cell body
(A-D) Confocal images of the neuronal cell body labelled for GFP-Hiw (green) in control (Elav-Gal4>UAS-GFP-Hiw) (A), Elav-Gal4>UAS-GFP-Hiw; UAS-Rab5^DN (B), Elav-Gal4>UAS-GFP-Hiw; UAS-Rab7^DN (C), and Elav-Gal4>UAS-GFP-Hiw; UAS-Rab11^DN (D). The scale bar in D represents 5 µm. (E) Histogram showing the number of GFP-Hiw puncta in control (Elav-Gal4>UAS-GFP-Hiw) (797 ± 20.1), Elav-Gal4>UAS-GFP-Hiw; UAS-Rab5^DN (755 ± 27.4), Elav-Gal4>UAS-GFP-Hiw; UAS-Rab7^DN (709 ± 24.8), Elav-Gal4>UAS-GFP-Hiw; UAS-Rab11^DN (291 ± 22.5). At least 8 VNCs of each genotype were used for the quantification. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001, ns: not significant (F-G’) Confocal images of the neuronal cell body co-labelled for GFP-Hiw (green) and Elav (magenta) in control (Elav-Gal4>UAS-GFP-Hiw) (F-F’), and Elav-Gal4>UAS-GFP-Hiw; Rab11^ex2/93Bi (G-G’). The scale bar in G’ represents 5 µm. (H) Histograms showing the number of GFP-Hiw puncta in the neuronal cell body of control (Elav-Gal4>UAS-GFP-Hiw) (716.1 ± 27.14), and Elav-Gal4>UAS-GFP-Hiw; Rab11^ex2/93Bi(160.5 ± 5.49). At least 12 VNCs of each genotype were used for the quantification. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using Student’s t-test. ***p<0.001. (I) Histogram showing the average size of the GFP-Hiw puncta in the neuronal cell body of control (Elav-Gal4>UAS-GFP-Hiw) (0.11 ± 0.01) and Elav-Gal4>UAS-GFP-Hiw; Rab11^ex2/93Bi (0.28 ± 0.01). At least 12 VNCs of each genotype were used for the quantification. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using Student’s t-test. ***p<0.001. (J) Western blot showing the level of GFP-Hiw in control (Elav-Gal4>UAS-GFP-Hiw) and Elav-Gal4>UAS-GFP-Hiw; Rab11^ex2/93Bi. Ran was used as a loading control. (K) Histogram showing the level of GFP-Hiw in control (Elav-Gal4>UAS-GFP-Hiw) (1.00 ± 0.00) and Elav-Gal4>UAS-GFP-Hiw; Rab11^ex2/93Bi (1.67 ± 0.05). Three independent Western blots were used for quantification. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using Student’s t-test. ***p<0.001. (L-N’’) Confocal images of third instar larval ventral nerve cord co-labelled for Wnd (magenta) and NC82 (green) in control (L-L’’), Rab11^ex2/93Bi(M-M’’), and AP2σ-^KG02457/AP2σ^ang7 (N-N’’). The scale bar in N’’ represents 10 µm. (O) Histogram showing the average fluorescence intensity of Wnd in control (0.55 ± 0.02), Rab11^ex2/93Bi (0.84 ± 0.03), and AP2σ-^KG02457/AP2σ^ang7 (1.03 ± 0.05). At least 10 VNCs of each genotype were used for the quantification. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001.

Endocytosis restricts JNK and BMP signaling through Rab11 in a parallel pathway
(A-D’’) Confocal images of muscle 6/7 NMJ showing the synaptic growth in control (A-A’’), Rab11^ex2/93Bi (B-B’’), Elav-Gal4>UAS-bsk^DN; Rab11^ex2/93Bi (C-C’’), and Elav-Gal4>UAS-kay^DN; Rab11^ex2/93Bi (D-D’’) immunolabeled for CSP (magenta) and HRP (green). The scale bar in D’’ represents 10 µm. (E) Histogram showing average bouton number from muscle 6/7 NMJ at A2 hemisegment in control (1.40 ± 0.07), Rab11^ex2/93Bi (3.70 ± 0.07), Elav-Gal4>UAS-bsk^DN; Rab11^ex2/93Bi (1.53 ± 0.0) and Elav-Gal4>UAS-kay^DN; Rab11^ex2/93Bi (1.64 ± 0.117). At least 8 NMJs of each genotype were used for the quantifications. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p<0.001; **p<0.01; *p<0.05 (F) Histogram showing the average bouton area from muscle 6/7 NMJ at A2 hemisegment in control (5.87 ± 0.87), Rab11^ex2/93Bi (2.02 ± 0.19), Elav-Gal4>UAS-bsk^DN; Rab11^ex2/93Bi (7.33 ± 0.88), and Elav-Gal4>UAS-kay^DN; Rab11^ex2/93Bi (6.91 ± 0.21). At least 8 NMJs of each genotype were used for the quantifications. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. **p<0.01; ns, not significant. (G) A proposed model for the regulation of synaptic growth signaling. Endocytosis regulates Rab11-positive recycling endosomes, which in turn modulate three partially independent pathways to attenuate synaptic growth. First, Rab11 regulates endosomal trafficking of Hiw; when trafficking is impaired, Hiw undergoes phase separation into large non-functional assemblies, which hyperactivates JNK signaling. Second, reduced Rab11 compromises autophagosome formation and autophagy, leading to elevated Hiw levels, which accumulate as phase-separated condensates in the endocytic mutants. Third, Rab11 regulates BMP receptor trafficking, and its reduction leads to increased pMad levels. These pathways suggest that endocytosis restricts synaptic growth by restraining BMP and JNK signaling in a parallel pathway mediated by Rab11-dependent endosomal trafficking, with Hiw phase separation as a key regulatory mechanism.

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