Ppk1 levels are not affected by decreasing dendrite arbor size but are reduced when dendrite length increases.

Images of ddaC neuron morphology in live 2nd instar larvae (72 h AEL). The dendritic membrane is marked by CD4::tdTomato, unless otherwise noted. Zoomed inset images show Ppk1::sfGFP in dendrite segments of neurons in live 3rd instar larvae (120 h AEL). (A) Representative images and quantification of control and Rpl22-RNAi-expressing neurons. Quantification, dendrite length: Student’s unpaired t-test (pRpl22-RNAi (13 neurons, 5 larvae). Dendrite length of Rpl22-RNAi-expressing neurons was quantified using Ppk1::sfGFP because the CD4::tdTomato signal was too dim to analyze in these mutants. Quantification, dendrite tip number: Mann-Whitney test (pRpl22-RNAi (13 neurons, 5 larvae). Quantification, Ppk1::sfGFP signal, dendrites: Student’s unpaired t-test (p = 0.8602); control (20 neurons, 9 larvae) v. Rpl22-RNAi (27 neurons, 14 larvae). (B) Representative images and quantification of control and EcR-DN-expressing neurons. Quantification, dendrite length: Student’s unpaired t-test (pEcR-DN (15 neurons, 7 larvae). Quantification, dendrite tip number: Student’s unpaired t-test (pEcR-DN (15 neurons, 7 larvae). Quantification, Ppk1::sfGFP signal, dendrites: Student’s unpaired t-test (p = 0.2318); control (24 neurons, 10 larvae) v. EcR-DN (23 neurons, 10 larvae). (C) Representative images and quantification of control neurons and neurons over-expressing Rac1 (Rac1 O/E). Quantification, dendrite length: Student’s unpaired t-test (p = 0.7618); control (15 neurons, 10 larvae) v. Rac1 O/E (14 neurons, 5 larvae). Quantification, dendrite tip number: Student’s unpaired t-test (pRac1 O/E (14 neurons, 5 larvae). Quantification, Ppk1::sfGFP signal, dendrites: Student’s unpaired t-test (p = 0.0791); control (20 neurons, 9 larvae) v. Rac1 O/E (20 neurons, 9 larvae). (D) Representative images and quantification of control neurons and neurons over-expressing PI3K (PI3K O/E). Quantification, dendrite length: Student’s unpaired t-test (p = 0.0001); control (12 neurons, 5 larvae) v. PI3K O/E (12 neurons, 6 larvae). Quantification, dendrite tip number: Student’s unpaired t-test (p = 0.0002); control (12 neurons, 5 larvae) v. PI3K O/E (12 neurons, 6 larvae). Quantification, Ppk1::sfGFP (AU), dendrites: Student’s unpaired t-test (pPI3K O/E (22 neurons, 13 larvae). Control genotype: w1118; ppk-Gal4. Experimental genotypes: ppk-Gal4 was used to express the indicated construct. Ppk-CD4::tdTomato and Ppk1::sfGFP included as indicated. Experiments to analyze the effects of Rpl22-RNAi and Rac1 over-expression were performed together; the controls for these experiments are the same. In the graphs, each data point represents a neuron, and data are plotted as mean ± SEM. n.s. = not significant (p>0.05), ***p = 0.001–0.0001, and ****p<0.0001. AU: arbitrary units. Scale bars, 50 μm.