Monensin Sensitive 1 Regulates Dendritic Arborization in Drosophila by Modulating Endocytic Flux

Abstract

Monensin Sensitive 1 (Mon1) is a component of the Mon1:Ccz1 complex that mediates Rab5 to Rab7 conversion in eukaryotic cells by serving as a guanine nucleotide exchange factor for Rab7 during vesicular trafficking. We find that Mon1 activity modulates the complexity of Class IV dendritic arborization (da) neurons during larval development. Loss of Mon1 function leads to an increase in arborization and complexity, while increased expression, leads to reduced arborization. The ability of Mon1 to influence dendritic development is possibly a function of its interactions with Rab family GTPases that are central players in vesicular trafficking. Earlier, these GTPases, specifically Rab1, Rab5, Rab10, and Rab11 have been shown to regulate dendritic arborization. We have conducted genetic epistasis experiments, by modulating the activity of Rab5, Rab7, and Rab11 in da neurons, in Mon1 mutants, and demonstrate that the ability of Mon1 to regulate arborization is possibly due to its effect on the recycling pathway. Dendritic branching is critical for proper connectivity and physiological function of the neuron. An understanding of regulatory elements, such as Mon1, as demonstrated in our study, is essential to understand neuronal function.

Keywords: Class IV neuron; Rab conversion; endocytic recycling; epistasis; flux.

FIGURE 1

Dmon1 modulates dendritic arborization in Class IV da -neurons. (A) A reporter (ppk-Gal4; ppk-GFP, diminutive ‘R’) line is used to visualize arborization in CIVda neurons at the third instar larval stage in Drosophila melanogaster. Sholl analysis (Image J) is used to calculate Ramification Index (R.I), which is then normalized, setting the ‘R/+’ at 100. The IMARIS software is used for neuron tracing and calculation of Dentritic area (D.A), Dendrite length (D.L) and Dendritic branch points (D.BP), with each parameter for the R/+ set at 100. n = 15 (neurons), N = 4 animals. Representative images are shown for this and other images (B–F). (B) Dmon1^Δ181/Dmon1^Δ181 larvae show enhanced arborization for Class IV arbors. n = 15, N = 4. (C) A Single copy of Dmon1 (Dmon1^Δ181/+) does not show any significant increase in arborization. n = 14 neurons, N = 4. (D) Dmon1^Δ181/Df(9062) increases arborization to the same extent as Dmon1^Δ181/Dmon1^Δ181. n = 15, N = 4. (E) UAS-Mon1:HA driven by ppk-Gal4 in Dmon1 ^Δ181 larvae, rescues the arborization defect in Dmon1^Δ181/Dmon1^Δ181 and Dmon1^Δ181/Df(9062) (Image not displayed) to near normal levels. n = 18, N = 5 and n = 9, N = 4 respectively. (F) Overexpression of Dmon1 in a wild-type background, reduces the branching (D.BP), R.I and D.L significantly. The reduction in D.A is less significant. n = 35, N = 8. (G–J) Quantitation of the extent of arborization in CIVDa using four parameters, R.I, D.A, D.L, and D.BP. Statistical analysis using Dunnet’s multiple comparison test using GraphPad Prism 7 with exact p-values listed in Supplementary Table S1. ns, not significant. ^*p < 0.05 and ^∗∗∗p < 0.001. Error bars represent standard error.

FIGURE 2

Rabs modulate dendritic arborization. (A) CIVda larvae imaged with the ‘wild-type’ reporter line (R/+: ppk-GFP, ppk-Gal4). Representative images are shown (B–I) for all experiments. (B–D) Rab5DN mutant expression or Rab5 knockdown using RNAi, reduces arborization (for Rab5 DN: n = 23, N = 4; for Rab5 RNAi: n = 40, N = 7) in Class IV neurons but Rab5CA mutant expression does not show any change (n = 18, N = 4), when compared to control R/+(n = 55, N = 5). (E–G) Rab7DN mutant expression, Rab7 knockdown using RNAi and Rab7CA mutant expression using ppk-Gal4, do not show any difference in arborization as compared to wild-type control (for Rab7DN: n = 35, N = 6; for Rab7 RNAi: n = 43, N = 7; for Rab7CA: n = 16, N = 4). (H,I) Rab11DN reduces arborization (n = 18, N = 4) when expressed in Class IV da neurons whereas Rab11CA mutant expression increases dendritic arbor complexity (n = 8, N = 3). (J,K) Quantitation of the extent of arborization in CIVDa using R.I and D.A. Values for D.L and D.BP are displayed in Supplementary Figure S2. ns, not significant. ^*p < 0.05 and ^∗∗∗p < 0.001. Error bars represent standard error. Statistical analysis using Dunnet’s multiple comparison test using GraphPad Prism 7 with exact p-values listed in Supplementary Table S1.

FIGURE 3

Mon1 interacts with Rabs to modulate dendritic arborization. (A,B) CIVda larvae imaged with the reporter line R/+ (n = 55, N = 7) and the Dmon1^Δ181/Dmon1^Δ181; R/+ (n = 38, N = 7). Representative images are shown (B–F) for all experiments. For (G–L), blue asterisk/n.s represents statistical comparison with wild-type (R/+) while red asterisk/n.s is a comparison with Dmon1^Δ181. (C,D) Rab5CA expression in Dmon1^Δ181 larvae shows significant reduction in parameters measured (R.I, D.A, D.L, D.BP) as compared to Dmon1^Δ181 (n = 30, N = 7). Rab5DN in Dmon1^Δ181 larvae shows reduction in arborization as compared to both Dmon1^Δ181 (n = 26, N = 5) and wild-type larvae (n = 26, N = 5). Rab7CA expression in Dmon1^Δ181 larvae shows reduction in arborization as compared to Dmon1^Δ181 (n = 20, N = 4) and increase with respect to the wild-type (n = 20, N = 4). Rab7 RNAi in Dmon1^Δ181 larave shows reduction in arborization as compared to Dmon1^Δ181 (n = 43, N = 7) and increase with respect to the wild-type (n = 43, N = 7). Images for these experiments are not shown but data is quantified in (H,K). (E,F) Rab11CA in Dmon1^Δ181 background shows reduction in arborization as compared to Dmon1^Δ181 larvae (n = 36, N = 6) and increase with respect to the wild-type larvae (n = 36, N = 6). Rab11 DN in Dmon1^Δ181 larvae shows reduction in arborization as compared to Dmon1^Δ181 (n = 19, N = 4) and decrease with respect to the control larvae (n = 19, N = 4). (G,L) Quantitation of the extent of arborization in CIVDa using R.I and D.A. Values for D.L and D.BP are displayed in Supplementary Figure S2. ns, not significant. ^*p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Error bars represent standard error. Statistical analysis using Dunnet’s multiple comparison test using GraphPad Prism 7 with exact p-values listed in Supplementary Table S1.

FIGURE 4

Mon1 levels may regulate vesicular flux through the recycling pathway. (A) Schematic shows the endocytic pathway branching at the early endosome, with vesicles entering either the degradative or recycling pathways. Rab5 marks early endosomes (EE), Rab7 marks late endosomes (LE) while Rab11 marks the recycling endosome (RE). The Mon1:Ccz complex acts as a GEF for conversion of EE to LE. (B) Effect of upregulation or downregulation of Rab or Mon1 activity on complexity of dendritic arborisation, as measured by change in R.I (red lines/arrows). R.I decreases with decrease in Rab5 and Rab11 activity as also with increase of Mon1 function. In contrast, R.I increases with decrease in Mon1 function and increase in Rab11 activity. Similar trends are seen in D.A, D.L, and D.BP. (C) A hypothetical model, that agrees with our data, is the requirement of endocytic recycling for increase in dendritic arborization. Thus, increase in RE endosomal flux may influence branching. This can be done directly by increasing RE by overexpressing Rab11, or indirectly by increasing the endocytic flux to the RE pathway, either by decreasing Mon1 function or by decreasing Rab5 activity. The proposed model relies on a minimal role for Rab7 in regulating R.I. Green arrows depict the increase in flux of vesicular trafficking in the RE pathway when Mon1 function is reduced.