Figure 1.
TnBVank1 expression causes the block of the transition from larval to pupal stage.
(A) Light micrographs of yw;h-Gal4 larva and pupa (control) and h-Gal4>TnBVank1 larvae at different days AED. The scale bar is 500 µm. (B) Larval length of different genotypes, at 96 h AED. Five larvae of each genotype were analyzed and as control we measured larval length of yw and h-Gal4 and UASp-TnBVank1 stocks and yw;h-Gal4. Graph represents mean ± standard deviation (s.d.); there is no significant (NS) length difference between h-Gal4>TnBVank1 (2680±83 µm) and yw;h-Gal4 larvae (2580±82 µm). (C) Larval length of h-Gal4>TnBVank1 increases during the extended larval life. Five h-Gal4>TnBVank1 larvae were measured at different days AED; values are the mean ± s.d. of three independent experiments. The mean values of h-Gal4>TnBVank1 larval length at four and eighteen days AED are shown above the bars.
Figure 2.
The expression of TnBVank1 in prothoracic gland affects the E biosynthesis.
(A) Ring gland includes the prothoracic gland (PG; yellow), the corpora allata (CA; orange) and the corpora cardiaca (CC; red). (B) The expression of the TnBVank1 gene is driven in the different ring gland compartments, highlighted in green, by three Gal4 drivers. P0206-Gal4>TnBVank1, expressed in PG and CA, causes the developmental arrest at the last larval stage; aug21-Gal4>TnBVank1 (CA) does not induce any developmental defects; phm-Gal4>TnBVank1 (PG) blocks the transition from larval to pupal stage. (C) Total 20E titer in five larvae of UASp-TnBVank1 stock (white bars), phm-Gal4/TM6B (grey bars) and phm-Gal4>TnBVank1 (black bars), at different time (hours AED). In the control stocks UASp-TnBVank1 and phm-Gal4/TM6B, the 20E peak which induces the pupariation is present at 120 h AED. Instead, this peak is absent in phm>TnBVank1 larvae at 120 h AED and during the extended larval life. Error bars represent s.d.; *** = p<0.0001 versus controls (UASp-TnBVank1 and phm-Gal4/TM6B). The mean values of total 20E at 120 h AED of different genotype larvae are shown above the bars. (D) Feeding TnBVank1 larvae with medium supplemented with 20E induces the pupariation (red), while TnBVank1 larvae fed with medium containing ethanol (EtOH) do not reach the pupal stage (green). Values are the mean ± s.d. of three independent experiments. The yw;phm-Gal4 larvae serve as background control (blue). Immunostaining with anti-Dib in yw;phm-Gal4 (E) and TnBVank1 (F) PG reveals that the expression of Dib is strongly reduced in all TnBVank1 PGs analyzed. Panels E,F are at the same magnification and the reference scale bar is 25 µm indicated in E.
Figure 3.
TnBVANK1 distribution in the PG cells and its effects on PG.
The immunolocalization of TnBVANK1 in PG cells (marked with mCD8::GFP, green), analyzed with anti-TnBVANK1 (cyan), shows its presence in stroke-shaped particles (A–C), which are distributed only in cytoplasm (nucleus is stained with Propidium Iodide, red) (B,C). (D) At five days AED, the PG of yw;phm-Gal4 larvae, marked with GFP, is significantly larger (+54%) than the TnBVank1 PG (E). (F) The graph represents the mean ± s.d.; 50 PGs were analyzed; *** = p<0.0001. (G) Measurement of PG cell area shows no difference between yw;phm-Gal4 and TnBVank1. 50 PGs were analyzed; NS: not significant. (H–J) Immunostaining with anti-Cleaved Caspase-3 (red) or TUNEL labeling (red) in PG cells marked with GFP. In the control yw;phm-Gal4 no caspase or TUNEL signal is detected (H,K), while in TnBVank1 PG few cells undergo apoptosis (I,J,L,M). PGs in panels A,D,E,H,I,K,L are at the same magnification and their scale bar is 25 µm and is showed in A. Scale bar in B and C is 5 µm and showed in B. Boxed regions are magnified in J and M and the reference scale bar is in B. (N) Larvae of yw;tub-Gal80ts/+;phm-Gal4/+ (Gal80ts-phm-Gal4) and UASp-TnBVank1;UASp-TnBVank1/tub-Gal80ts;phm-Gal4/+ (Gal80ts-TnBVank1) were raised at 21°C (cyan) for different time intervals, then shifted at 31°C (red) and dissected at 120 h AED. PG size from larvae incubated at 21°C until 96 h AED or until 72 h AED shows no significant (NS) differences from control larvae. PG size is strongly reduced in Gal80ts-TnBVank1 larvae incubated at 21°C until 48 h AED compared to PG from control larvae (*** = p<0.0001). Graph represents mean ± s.d.; 10 PGs were analyzed for each experiment.
Figure 4.
TnBVank1 PG cells have an altered cytoskeleton.
Phalloidin staining in control (A,B) and in TnBVank1 (C,D) PG cells. F-actin shows an altered distribution, characterized by thick masses of filaments in TnBVank1 PG cells. (E–H) α-tubulin-GFP fusion protein was expressed in yw;phm-Gal4 and TnBVank1 PG to investigate the microtubule network. Compared to control (E,F), in TnBVank1 the microtubule cytoskeleton is strongly affected and forms bundles (G,H). (I–L) Immunostaining with anti-Dynein heavy chain shows that, compared to control (I,J), in TnBVank1 PG cells the cortical localization of this protein is reduced and characterized by an evident dotted distribution (K,L). For each immunostaining we analyzed 60 PGs of five days AED larvae. PGs in panels A,C,E,G,I,K are at the same magnification and the reference scale bar is 25 µm and showed in A. Boxed regions are magnified in B,D,F,H,J,I and the reference scale bar 5 µm is indicated in B.
Figure 5.
TnBVank1 PG cells show lipids accumulation.
(A) In the control yw;phm-Gal4 there are few lipid droplets stained with Oil Red O, while in TnBVank1 cells several lipid droplets are detected (B,C). (E,F) In TnBVank1 there is also a sterol accumulation, shown by filipin staining, which is absent in control PG (D). 60 PGs were stained for each experiment. PGs in panels A,B,D,E are at the same magnification and the reference scale bar 50 µm is showed in A. Boxed regions are magnified in C,F and the reference scale bar is 5 µm indicated in C.
Figure 6.
TnBVANK1 disrupts the endocytic pathway in PG cells.
60 PGs stained for Rab5 (A,B), Rab11 (C,D) and Rab7 (E,F) in yw;phm-Gal4 and TnBVank1 larvae at five days AED. The distribution of endosomes marked with Rab5 (A,B) and Rab11 (C,D) is not affected by TnBVank1 expression, while a reduction in number was observed for late endosomes marked with Rab7 (E,F). All panels are at the same magnification and the reference scale bar is 5 µm showed in A.
Figure 7.
TnBVANK1 protein colocalizes with Hrs- and Alix-positive vesicles.
Confocal images of PG of yw;phm-Gal4 (A–C) and TnBVank1 (D–I) larvae stained for Hrs (cyan), Alix (red) and TnBVANK1 (green). In the control cells Alix (B) and Hrs (A) are widely distributed in the cytoplasm and their signals partially overlap (C). In TnBVank1 cells (D,F) a number of vesicles marked by Hrs have different shape compared to those present in controls (A,C). These modified vesicles show a strong colocalization with TnBVANK1 signal (E,F), demonstrating that TnBVANK1 protein is associated with Hrs-marked vesicles. In TnBVank1 cells (G) most of Alix-marked vesicles have different shape compared to those present in controls (B). Immunostaining with anti-Alix and anti-TnBVANK1 shows a strong colocalization of TnBVANK1 and Alix signals in the stroke-shaped vesicles (H). In these modified vesicles the Alix and the Hrs signals are both detected (I). PGs in all panels are at the same magnification and the reference scale bar is 5 µm indicated in A.
Figure 8.
Schematic overview showing the step in which TnBVANK1 affects the ecdysone biosynthesis in the PG cells.
EE: early endosomes, MVB: multivesicular bodies.