Journal: bioRxiv

Article Title: Filopodia-like Structures are Essential for Steroid Release

doi: 10.1101/2024.10.10.617560

Figure Lengend Snippet: (A) Inhibition of Rab11 results in an 80-hour delay in entering metamorphosis compared to the control. (B) Silencing of Vps25, Vps36, and shrub in PG cells resulted in a several-hour delay in entering metamorphosis compared to the control. (C, D) Images of phm>RNAi-Rab11 PG displaying a lack of microtubules (C) and βPS filopodia extension (D) in the sub-BM region. (E, F) ex vivo PG In a control situation, filopodia are visible when the cell expresses PLC-GFP (E). However, they are not observed anymore when Rab11 expression is down-regulated (F). (G, H) Comparison of control w 1118 PG (G) and phm>RNAi-Rab11 PG (H) showing F-actin (single section) and PTTH staining (maximum projection of a Z-stack) processed with a depth color code from a Fiji plugin. The YZ cuts allow us to see the position of the PTTH axons between the PG bilayer in the control PG (G), while when the recycling pathway is altered, PTTH axons are also observed below the BM (H). (I) Axon from a phm>RNAi-Rab11 PG at the sub-BM region. The PTTH staining co-localized with its receptor Torso, suggesting that these mislocalized synaptic buttons are functional. (J) A phm>RNAi-Rab11 PG at the sub-BM region shows the mislocalized synaptic button containing PTTH that represses βPS expression. (K) Image of a trol JO271#49 mutant PG Z sections reveals the loss of bilayer organization. (L) αTub85E staining in a trol JO271#49 mutant PG shows short microtubules in the sub-BM region. In the lateral plan, αTub85E staining looks normal. (M) Down-regulation of mys impedes microtubule extension. (N) Developmental timing of trol JO271#49 mutant vs w 1118 . The trol JO271#49 mutant enters pupariation 24 hours after the control line. White scale bar: 20 μm; yellow scale bar: 5 μm.

Article Snippet: Primary antibodies were diluted as follows: guinea pig monoclonal α-PTTH (1:400) , rabbit polyclonal α-Torso (1:200, this study), guinea pig polyclonal a-disembodied (1:400, this study), mouse monoclonal α-Arm (1:50, DSHB N27A1), rat monoclonal α-Ecad (1:50, DSHB DCAD2), α-Cora (1:50, DSHB C615.16), mouse monoclonal α-Dlg1 (1:50, DSHB 4F3), mouse monoclonal α-βPS (1:50, DSHB CF.6G11), rat monoclonal α-Ncad (1:50, DSHB DN-Ex #8), rat polyclonal α-Crb2.8 (1:300) , mouse monoclonal α-αTub85E (1:50, DSHB 12G10), rabbit polyclonal α-Trol (1:3500) , rabbit polyclonal α-Cg25c (1:1000) .

Techniques: Inhibition, Control, Ex Vivo, Expressing, Comparison, Staining, Functional Assay, Mutagenesis

Journal: bioRxiv

Article Title: Filopodia-like Structures are Essential for Steroid Release

doi: 10.1101/2024.10.10.617560

Figure Lengend Snippet: (A,A’) The PG is organized into a dorsal and ventral layer (A). 3D visualization of the ring gland (A’) composed of the PG (prothoracic gland), the corpora allata (CA) and the corpora cardiaca (CC). (B-B’’) Maximal projection of prothoracicotropic hormone (PTTH) staining (B) and two Z-sections revealing that PTTH projects its axons between the midline of the PG (B’, B’’). (C) Synaptic PTTH buttons reveal PTTH in the pre-synapse and Torso in the post-synapse. (D, D’) Expression of trol-GFP in the BM plan and in a lateral plan (D). The XZ section (D’) facilitates the observation of the trol-GFP dots. Those dots are CIVICs. (E) Z-sections reveal that the adherens junction markers Arm and ECad are located all around the PG cells. The septate junction markers and Dlg1 and the Cora and the βPS are also homogenously distributed in the PG cells. (F) Heatmap of Syt1-GFP in an XZ section of a cell revealed that the relative fluorescence intensity is higher in the sub-BM region compared to the lateral or midline membrane. The quantification is shown below. Scale bar: 20 μm. (G) Schematic representation of a PG dorsal layer and XZ cut.

Article Snippet: Primary antibodies were diluted as follows: guinea pig monoclonal α-PTTH (1:400) , rabbit polyclonal α-Torso (1:200, this study), guinea pig polyclonal a-disembodied (1:400, this study), mouse monoclonal α-Arm (1:50, DSHB N27A1), rat monoclonal α-Ecad (1:50, DSHB DCAD2), α-Cora (1:50, DSHB C615.16), mouse monoclonal α-Dlg1 (1:50, DSHB 4F3), mouse monoclonal α-βPS (1:50, DSHB CF.6G11), rat monoclonal α-Ncad (1:50, DSHB DN-Ex #8), rat polyclonal α-Crb2.8 (1:300) , mouse monoclonal α-αTub85E (1:50, DSHB 12G10), rabbit polyclonal α-Trol (1:3500) , rabbit polyclonal α-Cg25c (1:1000) .

Techniques: Staining, Expressing, Fluorescence, Membrane