Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells (2021)
Figure 4

Figure 4. Msps regulates microtubule assembly in the primary protrusion of qNSCs

A) Kymograph of EB-GFP comet movement in the primary protrusion of qNSCs from the control, mspsP18 and mspsP18/P with EB1-GFP expressed under grh-Gal4 at 6 h ALH. The horizontal arrow indicates the anterograde movement direction from the cell body to the tip of the primary protrusion in qNSCs. Both raw kymograph and kymograph with coloured lines were shown.

B) Quantification graph of fold changes in the number of EB1-GFP comets in the primary protrusion of qNSCs 6 h ALH from various genotypes compared with the control in (A). n=26 NSCs for control; n=18 NSCs for mspsP18; n=21 NSCs for mspsP18/P. ****p<0.0001.

C) Quantification graph of the percentage of retrograde EB1-GFP comets in the primary protrusion of qNSCs from the control, mspsP18 with EB1-GFP driven by grh-Gal4 at 6 h ALH. As EB1-GFP comets were almost lost in mspsP18, we were unable to capture adequate EB1-GFP comets for each individual brain to calculate the standard deviation. 38 and 41 qNSCs were analyzed in control and mspsP18, respectively. In control qNSCs, among total 383 EB1-GFP comets, 353 were anterograde and 30 were retrograde movements. In mspsP18 qNSCs, we only found 12 EB1-GFP comets with 6 anterograde and 6 retrograde movements.

D) Larval brains at 24 h ALH from control (grh-Gal4 UAS-CD8-GFP + UAS-nod-lacZ) and msps810,UAS-nod-lacZ under the control of grh-Gal4 UAS-CD8-GFP were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain were shown. Arrows point at the localization of Nod-lacZ in qNSCs.

E) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (D). n=55 NSCs for control; n=45 NSCs for msps810. ****p<0.0001.

F) Larval brains at 16 h ALH from control (grh-Gal4 UAS-CD8-GFP; UAS-dicer2 + UAS-nod-lacZ) and msps RNAi, UAS-nod-lacZ (VDRC#21982) with grh-Gal4 UAS-CD8-GFP; UAS-dicer2 were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain are shown. Arrows indicate the localization of Nod-lacZ in qNSCs.

G) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (F). n=60 NSCs for control; n=68 NSCs for msps RNAi. ****p<0.0001.

H) Larval brains at 24 h ALH from wild-type control (grh-Gal4>UAS-CD8-GFP), msps924, and mspsP18 expressing grh>CD8-GFP were labeled with Dpn and GFP. The arrow indicates the second protrusion in msps924.

I) Quantification graph of the thickness of the primary protrusion of qNSCs from wild-type, msps924, and mspsP18 expressing grh>CD8-GFP. The thickness was measured at the middle point of the primary protrusion. n=18 NSCs for control; n=52 NSCs for msps924; n=32 NSCs for mspsP18. **** p<0.0001.

J) Quantification graph of the length of primary protrusion in qNSCs in control (β-Gal RNAi) and msps RNAi (VDRC#21982) cells under the control of grh-Gal4 with UAS-CD8-GFP UAS-dicer2. n=40 NSCs for control; n=37 NSCs for msps RNAi. P=0.5257 (ns).

K) Quantification graph of qNSCs extending two major protrusions labeled grh>CD8-GFP in wild-type, msps924, and mspsP18 larval brains. n=18 NSCs for control; n=65 NSCs for msps924; n=71 NSCs for mspsP18.

L) Larval brains at 24 h ALH from control (grh-Gal4 UAS-CD8-GFP + UAS-nod-lacZ) and tacc74,UAS-nod-lacZ under the control of grh-Gal4 UAS-CD8-GFP; tacc59 were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain are shown.

M) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (L). n=51 NSCs for control; n=65 NSCs for tacc59/74. **p=0.0051.

Data information: in (B, C, E, G, I, J, K, M), data are presented as mean ± SD. In (B, I), statistical significances were determined by one-way ANOVA with multiple comparisons. In (J), statistical significance was determined by two-tailed Student's t-test. In (E, G, M), statistical significance was determined by two-way ANOVA with multiple comparisons. Scale bars: 10 µm.