Protein phosphatase PP2A regulates microtubule orientation and dendrite pruning in Drosophila

Abstract

Pruning that selectively eliminates inappropriate projections is crucial for sculpting neural circuits during development. During Drosophila metamorphosis, ddaC sensory neurons undergo dendrite-specific pruning in response to the steroid hormone ecdysone. However, the understanding of the molecular mechanisms underlying dendrite pruning remains incomplete. Here, we show that protein phosphatase 2A (PP2A) is required for dendrite pruning. The catalytic (Microtubule star/Mts), scaffolding (PP2A-29B), and two regulatory subunits (Widerborst/Wdb and Twins/Tws) play important roles in dendrite pruning. Functional analyses indicate that PP2A, via Wdb, facilitates the expression of Sox14 and Mical prior to dendrite pruning. Furthermore, PP2A, via Tws, governs the minus-end-out orientation of microtubules (MTs) in the dendrites. Moreover, the levels of Klp10A, a MT depolymerase, increase when PP2A is compromised. Attenuation of Klp10A fully rescues the MT orientation defects in mts or pp2a-29b RNAi ddaC neurons, suggesting that PP2A governs dendritic MT orientation by suppressing Klp10A levels and/or function. Taken together, this study sheds light on a novel function of PP2A in regulating dendrite pruning and dendritic MT polarity in sensory neurons.

Keywords: Klp10A; dendrite pruning; microtubule orientation; neuron; protein phosphatase.

Figure 1. Mts is required for dendrite pruning in ddaC neurons

1. A

   A Schematic representation of dendrite pruning in ddaC neurons.

2. B–F

   Live confocal images of ddaC neurons expressing UAS‐mCD8‐GFP driven by ppk‐Gal4 at WP and 16 h APF stages. Dendrites of ctrl RNAi (B), mts RNAi #1 (C), ctrl MARCM (D), mts ²⁹⁹ MARCM (E), and mts ²⁹⁹ rescue (F) ddaC neurons at WP and 16 h APF stages. Red arrowheads point to the ddaC somas.

3. G, H

   UAS‐mts‐dn ddaC neurons from animals in RU486‐induced condition (H) driven by GeneSwitch‐Gal4‐2295 exhibited normal arbors at WP stage and severe dendrite pruning defects at 16 h APF, compared to those in a non‐induced condition (G). Red arrowheads point to the ddaC somas.

4. I–K

   Quantification of number of primary and secondary dendrites attached to soma and percentage of severing defects at 16 h APF.

Data information: In (I–K), data are presented as mean ± SEM from three independent experiments. One‐way ANOVA with Bonferroni test (I) and two‐tailed Student's t‐test (K) were applied to determine statistical significance. ns, not significant; ***P < 0.001. The number of neurons (n) examined in each group is shown on the bars. Scale bars in (B–H) represent 50 μm.Source data are available online for this figure.

Figure EV1. Mts is essential for dendrite pruning and larval dendrite morphology in ddaC neurons

1. A

   Live confocal images of ddaC neurons expressing mCD8‐GFP driven by ppk‐Gal4 at WP and 16 h APF stages. Dendrites of mts ^xe2258 MARCM and mts ^xe2258 rescue ddaC neurons at WP and 16 h APF stages. Red arrowheads point to the ddaC somas.

2. B, C

   Live confocal images of ddaC neurons visualized by ppk‐Gal4‐driven mCD8::GFP expression at wL3 stage. ddaC neurons of mts ^xe2258 MARCM clone exhibited simplified dendrite arbors at WP stage. The rightest panel in B is the quantification of number of dendrite terminal and sholl analysis (C) for control and mts ^xe2258 MARCM ddaC clones.

Data information: In (B), data are presented as mean ± SEM from three independent experiments. ***P < 0.001 (two‐tailed Student's t‐test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A) represent 50 μm and (B) represent 20 μm. Source data are available online for this figure.

Figure 2. PP2A‐29B is required for dendrite pruning in ddaC neurons

1. A–E

   Live confocal images of ddaC neurons expressing UAS‐mCD8‐GFP driven by ppk‐Gal4 at WP and 16 h APF stages. Dendrites of ctrl RNAi (A), pp2a‐29b RNAi #1 (B), ctrl MARCM (C), pp2a‐29b ^rs MARCM (D), and pp2a‐29b ^rs rescue (E) ddaC neurons at WP and 16 h APF stages. Red arrowheads point to the ddaC somas.

2. F, G

   Quantification of number of primary and secondary dendrites attached to soma and percentage of severing defects at 16 h APF.

Data information: In (F‐G), data are presented as mean ± SEM from three independent experiments. ns, not significant; ***P < 0.001 (one‐way ANOVA with Bonferroni test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A–E) represent 50 μm.Source data are available online for this figure.

Figure 3. Two regulatory subunits of the PP2A, Wdb and Tws, are required for ddaC dendrite pruning

1. A–H

   Live confocal images of ddaC neurons expressing UAS‐mCD8‐GFP driven by ppk‐Gal4 at WP and 16 h APF stages. Dendrites of ctrl RNAi (A), wdb RNAi #1 driven by two copies of ppk‐Gal4 (B), tws RNAi #1 (C), ctrl MARCM (D), wdb ^dw MARCM (E), wdb ¹⁴ MARCM (F), tws ⁶⁰ MARCM (G), and tws ⁶⁰³ MARCM (H) ddaC neurons at WP and 16 h APF stages. Red arrowheads point to the ddaC somas.

2. I, J

   Quantification of number of primary and secondary dendrites attached to soma and percentage of severing defects at 16 h APF.

Data information: In (I–J), data are presented as mean ± SEM from three independent experiments. **P < 0.01; ***P < 0.001 (one‐way ANOVA with Bonferroni test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A–H) represent 50 μm.Source data are available online for this figure.

Figure EV2. Tws knockdown enhanced the pruning defects in wdb RNAi ddaC neurons

1. Live confocal images of ddaC neurons expressing mCD8‐GFP driven by ppk‐Gal4 at 16 h APF stage. Dendrites of ctrl RNAi, wdb RNAi #2, tws RNAi #2, wrd RNAi #1, wrd RNAi #2, wrd RNAi #3, pr72 RNAi #1, and pr72 RNAi #2 ddaC neurons at 16 h APF stage. Red arrowheads point to the ddaC somas. The numbers of neurons (n) examined for wrd and pr72 are shown on the panels.

2. Live confocal images of ddaC neurons expressing mCD8‐GFP driven by ppk‐Gal4 at 16 h APF stage. Dendrites of wdb RNAi + ctrl RNAi, tws RNAi + ctrl RNAi, and wdb RNAi + tws RNAi ddaC neurons at 16 h APF stage. Red arrowheads point to the ddaC somas. Quantification of number of primary and secondary dendrites attached to soma and percentage of severing defects in these genotypes at 16 h APF (bottom panels).

Data information: In (B), data are presented as mean ± SEM from three independent experiments. ***P < 0.001 (one‐way ANOVA with Bonferroni test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A, B) represent 50 μm.Source data are available online for this figure.

Figure 4. PP2A is required to regulate ecdysone signaling during dendrite pruning

1. A–O

   Confocal images of control (A–C), mts ^xe2258 (D–F), pp2a‐29b ^rs (G–I), wdb ^dw (J–L), and tws ⁶⁰ (M–O), MARCM ddaC clones that were immunostained for EcR‐B1 (A, D, G, J, M) and Sox14 (B, E, H, K, N), and Mical (C, F, I, L, O) at WP stage. ddaC somas are labeled by dashed lines, ddaE by asterisks. ddaC neurons were identified by ppk‐Gal4‐driven mCD8::GFP (green channel) expression, as shown at the top right corner.

2. P–R

   Quantitative analyses of normalized EcR‐B1, Sox14, and Mical fluorescence intensities in ddaC neurons.

Data information: In (P–R), data are presented as mean ± SEM from three independent experiments. ns, not significant; *P < 0.05; ***P < 0.001 (one‐way ANOVA with Bonferroni test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A–O) represent 10 μm.Source data are available online for this figure.

Figure 5. Mical overexpression significantly suppressed the dendrite pruning defects in PP2A mutant neurons

1. A–I

   Live confocal images of ddaC neurons expressing UAS‐mCD8‐GFP driven by ppk‐Gal4 at WP and 16 h APF stages. Dendrites of ctrl MARCM (A), mts ²⁹⁹ MARCM (B), UAS‐Mical + mts ²⁹⁹ MARCM (C), ctrl MARCM (D), pp2a‐29b ^rs MARCM (E), UAS‐Mical + pp2a‐29b ^rs MARCM (F), ctrl RNAi (G), wdb RNAi + UAS‐Control (H), and wdb RNAi + UAS‐Mical (I) ddaC neurons at 16 h APF stage. Red arrowheads point to the ddaC somas.

2. J, K

   Quantification of number of primary and secondary dendrites attached to soma and percentage of severing defects at 16 h APF.

Data information: In (J–K), data are presented as mean ± SEM from three independent experiments. *P < 0.05; ***P < 0.001 (two‐tailed Student's t‐test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A–I) represent 50 μm.Source data are available online for this figure.

Figure EV3. The distribution of F‐actin appeared to be largely normal between wild‐type and mts/pp2a‐29b RNAi ddaC neurons at wL3

1. Confocal images of ctrl RNAi, mts RNAi, and pp2a‐29b RNAi ddaC neurons immunostained for phalloidin at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons were identified by ppk‐Gal4‐driven mCD8::GFP (green channel) expression. ddaC somas are labeled by dashed lines, and ddaE somas are marked by asterisks.

2. Quantitative analysis of normalized phalloidin fluorescence intensities of ddaC somas.

Data information: In (B), data are presented as mean ± SEM from three independent experiments. ns, not significant (one‐way ANOVA with Bonferroni test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A) represent 10 μm.Source data are available online for this figure.

Figure 6. PP2A is essential for proper distribution of dendritic and axonal MT markers

1. A–E

   Confocal images of ddaC neurons at the wandering 3^rd instar (wL3) stage immunostained for anti‐β‐galactosidase. Nod‐β‐gal signals were localized in the dendrites of the ctrl RNAi (A) and wdb ^dw (E) mutant ddaC neurons; however, Nod‐β‐gal levels were strongly reduced in the dendrites and accumulated in the somas in mts RNAi (B), pp2a‐29b RNAi (C), and tws RNAi (D) ddaC neurons. ddaC somas are marked by asterisks, axons by arrows, and dendrites used for analyzing by curly brackets.

2. F–J

   Kin‐β‐gal was mislocalized to the dendrites in mts RNAi (G), pp2a‐29b RNAi (H), tws RNAi (I), and wdb ^dw/ wdb ¹⁴ (J) mutant ddaC neurons, compared to the ctrl RNAi neurons (F), while the localization of Kin‐β‐gal in wdb ^dw/ wdb ¹⁴ mutant (J) is normal. ddaC somas are marked by asterisks, axons by arrows, and white arrowheads point to dendritic Kin‐β‐gal signals.

3. K

   Quantification of normalized Nod‐β‐gal fluorescence intensity in dendrites ofddaC neurons.

4. L, M

   Quantification of the percentage of neurons with defective Nod‐β‐gal and Kin‐β‐gal distribution in ddaC neurons.

Data information: In (K–M), data are presented as mean ± SEM from three independent experiments. ns, not significant; ***P < 0.001 (one‐way ANOVA with Bonferroni test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A–J) represent 10 μm.Source data are available online for this figure.

Figure 7. PP2A is required for uniform minus‐end‐out orientation of dendritic MTs in ddaC neurons

1. A–D

   Representative kymographs depicting the movement patterns of EB1 comets in the proximal dendrites of ddaC neurons at 96 h AEL. In control RNAi (A) ddaC dendrites, EB1‐GFP comets predominantly moved toward the somas (retrograde). However, in mts RNAi (B), pp2a‐29b RNAi (C), and tws RNAi (D) ddaC dendrite branches, some EB1‐GFP comets moved away from the somas (anterograde).

2. E–H

   Quantitative analyses of the percentages of anterograde EB1 comets in each neuron imaged (E), the average numbers of EB1‐GFP comets within 30 μm dendrite in 3 min (F), the average comet track length of each neuron (G), and the average comet speed of each neuron (H).

3. I, J

   Representative kymographs depicting the movement patterns of EB1 comets in the proximal dendrites of ddaC neurons at 96 h AEL. In both control (I) and wdb ^dw /wdb ¹⁴ mutant (J) ddaC dendrites, EB1‐GFP comets predominantly moved toward the somas (retrograde).

4. K–N

   Quantitative analyses of the percentages of anterograde EB1 comets in each neuron imaged (K), the average numbers of EB1‐GFP comets within 30 μm dendrite in 3 min (L), the average comet track length of each neuron (M), and the average comet speed of each neuron (N).

Data information: In (E–H) and (K–N), data are presented as mean ± SEM from three independent experiments. ns, not significant; *P < 0.05; ***P < 0.001 (E–H, one‐way ANOVA with Bonferroni test; K–N, two‐tailed Student's t‐test). The number of neurons (n) examined in each group is shown on the bars. Horizontal arrow indicates the direction toward the somas, and vertical arrow indicates that each movie was taken for 3 min. Scale bars in (A, I) represent 10 μm.Source data are available online for this figure.

Figure 8. PP2A regulates dendritic MT orientation through suppressing Klp10A levels

1. A

   Confocal images of ctrl RNAi, mts RNAi, and pp2a‐29b RNAi ddaC neurons immunostained for Klp10A at wL3 stages. ddaC somas are labeled by dashed lines; ddaE somas are marked by asterisks.

2. B

   Quantitative analysis of normalized Klp10A fluorescence intensities of ddaC somas.

3. C–H

   Representative kymographs depicting the movement patterns of EB1 comets in the proximal dendrites of ddaC neurons at 96 h AEL in (C) mts RNAi + ctrl RNAi and mts RNAi + klp10a RNAi; (E) pp2a‐29b RNAi + ctrl RNAi and pp2a‐29b RNAi + klp10a RNAi; and (G) tws RNAi + control RNAi and tws RNAi + klp10a RNAi. (D, F, H) Quantitative analyses of the percentages of anterograde EB1 comets in ddaC neurons.

4. I

   A possible model. PP2A, via Wdb, regulates ddaC dendritepruning through the expression of Sox14 and Mical, whereas PP2A, via Tws, regulates dendritic MT polarity via suppressing the Klp10A levels.

Data information: In (B, D, F, H), data are presented as mean ± SEM from three independent experiments. ns, not significant; **P < 0.01; ***P < 0.001 (B, one‐way ANOVA with Bonferroni test; D, F, H, two‐tailed Student's t‐test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A, C, E, G) represent 10 μm. (C, E, G) Horizontal arrow indicates the direction toward the somas, and vertical arrow indicates that each movie was taken for 3 min.Source data are available online for this figure.

Figure EV4. PP2A is required to suppress the levels of Klp10A in class I ddaD/E neurons

1. A–H

   Confocal images of ddaC neurons of (A) ctrl RNAi, mts RNAi + control RNAi and mts RNAi + klp10a RNAi; (C) ctrl RNAi, mts RNAi and pp2a‐29b RNAi; (E) ctrl MARCM and wdb ^dw MARCM; and (G) ctrl RNAi and mical RNAi. All were immunostained for Klp10A at wL3 stage. ddaC (A, E, G) and ddaD/E (C) somas are labeled by dashed lines, and ddaE somas (A, E, G) are marked by asterisks. ddaC (A, E, G) and ddaD/E (C) neurons were identified by the mCD8::GFP expression driven by ppk‐Gal4 and Gal4 ^2‐21‐driven, respectively. (B, D, F, H) Quantitative analysis of normalized Klp10A fluorescence intensities of ddaC or ddaD somas.

Data information: In (B, D, F, H), data are presented as mean ± SEM from three independent experiments. ns, not significant; ***P < 0.001 (B, D, one‐way ANOVA with Bonferroni test; F, H, two‐tailed Student's t‐test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A, C, E, G) represent 10 μm.Source data are available online for this figure.

Figure EV5. PP2A regulates dendrite pruning partially through suppressing the Klp10A level

1. Live confocal images of ddaC neurons expressing mCD8‐GFP driven by ppk‐Gal4 at 16 h APF stage. Dendrites of mts RNAi + Ctrl RNAi, mts RNAi + klp10a RNAi, pp2a‐29b RNAi + Ctrl RNAi, and pp2a‐29b RNAi + klp10a RNAi ddaC neurons at 16 h APF stage. Red arrowheads point to the ddaC somas. Quantification of number of primary and secondary dendrites attached to soma and percentage of severing defects at 16 h APF (rightest panels).

2. Confocal images of ddaC neurons of wild‐type, mts RNAi + Ctrl RNAi, mts RNAi + klp10a RNAi, wild‐type, pp2a‐29b RNAi + Ctrl RNAi, and pp2a‐29b RNAi + klp10a RNAi that were immunostained for Mical at WP stage. Quantitative analyses of normalized Mical fluorescence (rightest panels). ddaC somas are labeled by dashed lines, and ddaE somas are marked by asterisks.

Data information: In (A, B), data are presented as mean ± SEM from three independent experiments. ns, not significant; **P < 0.01 (two‐tailed Student's t‐test); ***P < 0.001 (one‐way ANOVA with Bonferroni test). The number of neurons (n) examined in each group is shown on the bars. Scale bars in (A) and (B) represent 50 and 10 μm, respectively.Source data are available online for this figure.