Fig 1.
Small heat shock proteins modulate synapses during Drosophila development.
Synapses quantification screening with sHsps genetic tools under D42 driver expression. (A) Synapses modulation were detected by sHsp20 RNAi (sHsp20↓), sHsp22 RNAi (sHsp22↓), sHsp23 (sHsp23↓), sHsp26 RNAi (Hsp26↓), sHsp27 RNAi (Hsp27↓), Hsp40 RNAi (Hsp40↓), Hsp90 RNAi (Hsp90↓), (B) UAS.sHsp23 (Hsp23↑) UAS.sHsp26 (Hsp26↑) and UAS.Hsp70 (Hsp70↑) samples. One‐way ANOVA test with Dunn's multiple comparisons post‐test. *p value ≤ .05; ** p value ≤ .01; *** p value ≤ .001. p value > .05 were not considered significant. Error bars show S.D. (C) Diagram of sHsp23 interactome and (D) diagram of sHsp26 interactome form http://flybi.hms.harvard.edu/results.php.
Fig 2.
sHSP23 and sHSP26 colocalize in CNS.
(A-F) Confocal microscopy images of 3rd instar Drosophila larval brain and NMJs. (A) sHSP23 is labeled with anti-GFP antibody driven by D42-Gal4 to visualize its expression in brain regions (magenta). Scale bar size 100 um. (B) sHSP26 is stained with anti-sHSP23 (green). (A`-B`) Magnification images of larval brain. Arrows indicate neuroblast, arrowheads indicate ganglion mother cells and asterisk indicate neurons where sHSP23 and sHSP26 colocalize in the cytoplasm. Scale bar size 100 um. (C-F) sHSP26 is labeled with anti-GFP antibody driven by D42-Gal4 to visualize its expression in NMJ (red), sHSP23 is stained with anti-sHSP23 (green) and neuronal membrane is detected with anti-HRP staining (magenta). Scale bar size 50 um (C`-F`) Magnification images of synaptic boutons in NMJ. (G) Co-Immunoprecipitation assay membrane revealed with sHSP26 (green, arrow) and sHSP23 (red) antibodies in control samples. Fly heads were lysed in immunoprecipitation lysis buffer and incubated with protein A/G agarose beads previously treated with sHSP23 or sHSP26 antibody and IgG antibody as a control. The samples were prepared for western blot analysis. The antibody-protein interaction is visualized by chemoluminescence. Molecular weights are indicated in all the membrane images. * Unknown/unspecific band (H) sHSP23 and sHSP26 interaction diagram.
Fig 3.
Pkm does not affect to sHSP23-sHSP26 interaction.
(A) Quantification of synapse active zones in the NMJ is shown for the knockdown of all candidate genes genotypes: CG43755 RNAi (CG43755↓), CG11534 RNAi (CG11534↓) and pkm RNAi (pkm↓). One‐way ANOVA test with Bonferroni post‐test* P<0.05. Error bars show S.D. (B) pkm contains a EcKinase like (Ecdysteroid kinase-like) domain between 257–545 aa sequence and a CHK_kinase like (Choline kinase-like) domain between 346–543 aa sequence. (C) Diagram of Pkm interactome. Pkm physically interacts with sHSP23 and sHSP26. (D) Co-immunoprecipitation assay membrane revealed with sHSP23 antibody in control and pkm RNAi samples. Molecular weights are indicated.
Fig 4.
sHSPs amount is regulated by the novel candidate gene pkm.
(A) qPCR assay of pkm RNAi sample measuring mRNA expression fold change of 3rd instar larval of pkm, sHsp23, sHsp26 expression and Cat as positive control, normalized with Rp49 as a control. (B) Western blot assay of control and pkm RNAi (pkm↓) samples stained against sHSP23 and sHSP26. We used three RNAi tools to confirm the protein amount changes under pkm downregulation condition. pkm RNAi 2 was selected due to its efficacy. Tubulin was used as a control. (C) Mean Intensity sHSP23 and sHSP26 signal are shown for control and pkm RNAi (pkm↓) samples. Unpaired T-test Welch´s correction* P<0.05. Error bars show S.D. (D) Quantification of synapse active zones in the NMJ is shown for the combination of sHsp23 and pkm, sHsp26 downregulation under D42 driver expression. (E) Synapse number quantification in NMJs after sHsp26 upregulation and pkm downregulation. Unpaired T-test Mann Whitney post-test * p value<0.05; p value > .05 were not considered significant. Error bars show S.D.
Fig 5.
Pkm activity is restricted by sHsps expression.
Quantification of synapse active zones in the NMJ is shown for the combination of sHsps expression and pkm downregulation under D42 driver expression: (A) pkm RNAi (pkm↓), UAS.sHsp23; UAS.sHsps26 (sHsp23↑; sHsp26↑), UAS.sHsp23; UAS.sHsps26/pkm RNAi (sHsp23↑; sHsp26↑/pkm↓), (B) UAS.sHsp23 RNAi; UAS.sHsps26 RNAi (sHsp23↓; sHsp26↓), UAS.sHsp23 RNAi; UAS.sHsps26 RNAi /pkm RNAi (sHsp23↓; sHsp26↓/pkm↓). One‐way ANOVA test with Bonferroni post‐test. *p value ≤ .05; ** p value ≤ .01; *** p value ≤ .001. p value > .05 were not considered significant. Error bars show S.D. (C) Summary table for the combination of sHsps expression and pkm downregulation.
Fig 6.
sHSPs contribute to neuronal activity GFP signal.
(A) CalexA system labels neuronal activity based on calcium/NFAT signaling after a neuronal action potential and the two binary expression systems UAS/Gal4 and LexA/LexAop. The accumulation of calcium activates calcineurin that dephosphorylates NFAT that are imported to the nucleus. NFAT binds to LexAop sequence and induces the expression of GFP reporter gene that correlates with neuronal activity. (B-D) Confocal microscopy images of 3rd instar Drosophila larval ventral nerve cord of (B) control, (C) UAS.sHsp23; UAS.sHsps26 (sHsp23↑; sHsp26↑), (D) pkm RNAi (pkm↓) samples. (E) GFP mean intensity signal quantification is shown for sHsps expression and pkm downregulation under D42 driver expression: UAS.sHsp23; UAS.sHsps26 (sHsp23↑; sHsp26↑), pkm RNAi (pkm↓), One‐way ANOVA test with Bonferroni post‐test.; *** p value = 0.002. **** p value<0,0001;. p value > .05 were not considered significant. Error bars show S.D.