EMBO/SourceData huggingface dataset:
Figure 2UCP1 and PPARγ protein levels in BAT and inguinal WAT (iWAT) from mice in (E).I,J. Mice supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal (I) and CL316,243-stimulated (J) states. n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.
Figure 3Rectal temperature in mice of (A) during cold exposure. n=5 mice each group.BAT and inguinal WAT (iWAT) gene expression analysis in mice of (A).Hematoxylin and eosin staining of BAT, inguinal WAT (iWAT) and epididymal WAT (eWAT) of mice in (A).
Figure 4Glucose tolerance test was performed after a high-fat diet feeding for 17 weeks in mice of (A). n=5 mice each group.   Serum insulin levels in mice of (A). n=5 mice each group.   Serum triglyceride levels in mice of (A). n=5 mice each group.   Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.BAT and inguinal WAT (iWAT) UCP1 and PPARγ protein levels in mice of (A).
Figure 5Heat map of metabolite relative levels in brown fat cells treated with or without 1mM asparagine.Immunoblot detection of the glucose transporters, glycolysis and TCA key enzymes in primary brown fat cell line Asparagine was supplemented to cells during differentiation.Immunoblot detection of the glucose transporters, glycolysis and TCA key enzymes in immortalized brown fat cell line (E), BAT from mice fed asparagine (F) and injected asparaginase (G). Asparagine was supplemented to cells during differentiation.
Figure 6Intracellular abundance of 13C-isotopologue metabolites downstream of glycolysis in brown adipocytes. n=5 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.C,D. Quantification of glucose (C) and lactate (D) concentration from media. The media was collected after incubated with mature brown adipocytes for 48 h. n=3 independent experiments in (C), n=6 independent experiments in (D). Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.
Figure 7Quantification of intracellular amino acid levels in brown fat cells (day 6) treated with or without 1mM asparagine. n=4 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.B-D. mTORC1 pathway activity was analyzed by immunoblotting for the phosphorylation of 4E-BP1 and S6K in brown adipocytes (B), BAT from mice fed asparagine (C) or asparaginase (D). (B) Asparagine was added to cultured media from day 4 to day 6.Brown preadipocytes were induced to differentiation with or without 1mM asparagine. Rapamycin (75nM) was added to cells for 48h. Then western blot detected protein expression on day 6.Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation. Protein expression analysis was performed on day 6.
Figure 1F. Dopaminergic neuronal cultures from control lines and glucocerebrosidase (GBA) N370S PD patients were immunoblotted for the presence of LAG3. No band for LAG3 could be observed in neurons.G. Using high power, high resolution laser scanning confocal microscopy, no human LAG3 signal could be detected in human neurons (Auto-hLAG3 transduced, DOX OFF) by two different anti-human LAG3 antibodies (17B4 and D2G40; left panel and zoomed-in insets) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 (DOX ON; right panel and zoomed-in insets). Scale bars 25 µm.H. Human brain homogenates from autopsy material were immunoblotted for the presence of LAG3. No band for LAG3 could be evidenced in any of the brain homogenates. Control samples (tonsils and activated human T cells) show expected bands.
Figure 2B. Mixed neuroglial, neuron-enriched, and glial cortical cultures were assayed for the presence of LAG3 where no band could be found. Activated T cells display band at expected size.D. Using high power, high resolution laser scanning confocal microscopy, no mouse LAG3 signal could be detected in murine primary neuronal cultures (Auto-mLAG3 transduced, DOX OFF and non-transduced neurons) using anti-mouse LAG3 antibody (MABF954) whereas LAG3 was clearly detected in primary neurons induced to express mLAG3 (Auto-mLAG3 transduced, DOX ON). Scale bars 20 µm.E. RT-qPCR-derived CT values to measure the cDNA copy number. Mixed neuroglial cultures, glial cultures, and neuron-enriched cultures show transcript levels that are close to indistinguishable to those from LAG3‑/‑ BH. Conversely, around 1600 transcripts/ng of RNA were counted in activated T cells.
Figure 3A. and B. Binding of ASC filaments (A) or α-synuclein PFFs (B) to multiple proteins. Serial dilutions of ASC filaments and α-synuclein PFFs were performed and bound fractions were detected using anti-ASC or anti-α-synuclein (MJFR1) antibodies. ASC filaments did not interact with any of the proteins (A). Conversely, α-synuclein PFFs strongly bound LAG3, CD4, and MTBD tau and showed moderate binding properties to PrPC, APOE3, and TDP-43. No binding between α-synuclein PFFs and nAra h 2 or BSA could be detected. Values represent means ± SD of technical duplicates.C. and D. Diffusional sizing of α-synuclein PFFs with fluorescently conjugated LAG3, CD4, BSA, and MJFR1 α-synuclein antibody (C) and of fluorescently labelled LAG3 with monomeric α-synuclein, α-synuclein PFFs, FGL1, and Relatlimab anti-LAG3 antibody (D). An increase in the hydrodynamic radius (Rh) of α-synuclein PFFs is seen with increasing concentrations for MJFR1 but not for LAG3 or any of the control proteins, also not at a lower scale (see insert) (C). Similarly, the hydrodynamic radius of labelled LAG3 increases with higher concentrations of Relatlimab and FGL1 but does not change with α-synuclein PFFs or monomeric α-synuclein (D). Values are given as means ± SD of technical triplicates.E. and F. Co-immunoprecipitation of LAG3 and subsequent immunoblotting with anti α-synuclein antibody (MJFR1) (E) and co-immunoprecipitation of α-synuclein (monomeric or PFFs) and subsequent immunoblotting with anti-LAG3 antibody (D2G40) (F). Using the anti-α-synuclein antibody for pulldown resulted in LAG3 bands with both monomeric as well as fibrillar α-synuclein. The usage of anti-LAG3 for pulldown led to the specific detection of α-synuclein PFFs, although the PFF fraction predominates also in the input and supernatant. Bands for α-synuclein (E) and LAG3 (F) are indicated by arrows.
Figure 4Human neural cultures transduced by Auto-hLAG3 were treated by α-synuclein PFFs 4 days post LAG3 expression induction by DOX and kept in culture for 2 or 4 weeks. Both transgenic (hLAG3 D2G40-positive in (A) and 17B4-positive in (B)) and non-transduced, wild-type neurons (selected neurons in zoomed-in insets) propagated α-synuclein and developed characteristic pS129-positive (81A-positive in (A) and EP1536Y-positive in (B)) α-synuclein aggregates. Scale bars 25 µm. Trained ilastik algorithms were used to segment pixels of 81A, EP1536Y and MAP2 stainings imaged by high-content wide-field microscope, which were used to quantify the signal of 81A-positive (C) and EP1536Y-positive (D) α-synuclein aggregates expressed as % of MAP2-positive area. Almost the entire wells (182 fields per well) were imaged for every condition and replicate and each datapoint in the plot represents the entire well. Error bars indicate mean ± SD of biological replicates (duplicates). For few conditions, unicates were used, shown as one dot. One-way ANOVA followed by Tukey's multiple comparison test demonstrated that neurons that did not express LAG3 (DOX OFF) showed no difference in α-synuclein propagation when compared to LAG3-expressing neurons (DOX ON) as demonstrated by two different pS129 α-synuclein antibodies (p=0.9998 for 81A at 2 weeks and p=0.2522 at 4 weeks; p=0.9986 for EP1536Y at 2 weeks and p=0.3042 at 4 weeks).
Figure 5A. Survival curves of A53T α-synuclein TG mice, with LAG3 knockout, heterozygous LAG3 expression, or LAG3 WT. Median survival was shortest for α-synuclein TG LAG3‑/‑ at 239 d, followed by α-synuclein TG LAG3+/- at 260 d and α-synuclein TG LAG3+/+ at 268 d. The survivals of ASYNA53T LAG3‑/‑, LAG3+/- and LAG3+/+ mice were similar (Mantel-Cox log-rank test, p-value = 0.165). Mice were euthanized and dissected in the late-stage symptomatic phase, which would inevitably result in death within a week. None of the control LAG3‑/‑ α-synuclein WT mice died over the course of the experiment.B. Immunostaining for hyperphosphorylated α-synuclein aggregates (pS129) and aggregated protein (Thioflavin S) in sections of end-stage symptomatic mice; no obvious difference in terms of staining pattern or number of stained cells were apparent in midbrain or brain stem. Scale bars represent 1 mm (sagittal section) and 100 µm (panel).C. Immunofluorescence staining and quantification of pS129-positive α-synuclein inclusions in hippocampal slice cultures (HSCs) at 5 weeks post-seeding with 350 µM PFF. Scale bars represent 500 µm (overview), and 100 µm (insets). Shown is mean ± SEM and individual values are shown; n = 6 cultures per group; two-way-ANOVA revealed for LAG3 F(1, 20) = 0.0294; p = 0.8656; A53T F(1, 20) = 36.42, p < 0.0001; interaction F(1, 20) = 0.0062, p = 0.9382. Bonferroni's correction for multiple comparisons revealed p = 0.9973 for LAG3 WT vs. KO in A53T TG and p > 0.9808 for LAG3 WT vs. KO in A53T WT.
Figure 1(A) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), dNmnat (DVGlut-Gal4 > UAS-dNmnat), or Wnd (DVGlut-Gal4 > UAS-Wnd).(B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 13 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=36, F=12.66, p<0.0001. UAS-RFP vs UAS-dNmnat p < 0.0001 (****), UAS-RFP vs UAS-Wnd p = 0.0093 (**)].]. (C) Quantification of mean (±SEM) mEPSP amplitudes, in which 75 consecutive spontaneous events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=11.66, p = 0.0001. UAS-RFP vs UAS-dNmnat p=0.994 (NS), UAS-RFP vs UAS-Wnd p = 0.0003 (***)].(D) Quantification of quantal content, which was calculated individually per cell by dividing the mean EPSP amplitude by the mean mEPSP amplitude, and then averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=9.286, p = 0.0006. UAS-RFP vs UAS-dNmnat p=0.0015 (**), UAS-RFP vs UAS-Wnd p = 0.0003 (***)].]. (E) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in the electrophysiology experiments in A-D. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red).(F) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [UAS-RFP n = 23, UAS-dNmnat n = 21, UAS-Wnd n= 20 NMJs. 1-way ANOVA w/Tukey's multiple comparisons, DF=63, F=68.31, p < 0.0001. UAS-RFP vs UAS-dNmnat p=0.9818 (NS), UAS-RFP vs UAS-Wnd p < 0.0001(****)].
Figure 2(A) Representative EPSP and mEPSP physiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing RNAis to knockdown either dNmnat (hiwND8;Elav-Gal4 > UAS-dNmnat-RNAi [#29402] ) or Wnd (hiwND8;Elav-Gal4 > UAS-Wnd-RNAi [#25396]).(B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=39, F=14.02, p<0.0001. WT vs hiw p = 0.0012 (**), WT vs hiw > Wnd-RNAi p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p = 0.9880 (NS)].(C) Quantification of quantal content (±SEM). [WT n = 9 cells, hiw n = 9 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F= 2.508, p<0.0001. WT vs hiw p = 0.0361 (*), WT vs hiw > Wnd-RNAi p = 0.0001 (***), WT vs hiw > dNmnat-RNAi p = 0.9889 (NS)].(D) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels A-C. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red).(E) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 23, hiw n = 21 , hiw > Wnd-RNAi n= 16, hiw > dNmnat-RNAi n=19. 1-way ANOVA w/Tukey's multiple comparisons, DF=78, F=169.1, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Wnd-RNAi p = 0.9988 (NS), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.9695 (NS)].
Figure 3(A) Representative images of immunostained larval NMJs and active zones from WT, hiw mutant, and hiw mutant driving an RNAi against dNmnat [#29402]. In the top 3 panels, greyscale images of entire terminal were acquired by staining for nerve marker HRP. Presynaptic Brp puncta (green) and postsynaptic dGluRIII clusters (magenta) were imaged to quantify N in each genotype.(B) Quantification of NMJ synaptic terminal surface area in each genotype. [WT n = 14, hiw n = 12 , hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=22.427, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.2907 (NS)].(C) Quantification of BRP puncta per NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=2.854, p = 0.0711. WT vs hiw p = 0.0573 (NS), WT vs hiw > dNmnat-RNAi p = 0.4529 (NS), hiw vs hiw > dNmnat-RNAi p = 0.5030 (NS)].(D) Quantification of percentage of Brp puncta that were apposed to a dGluRIII clusters at each NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=2.093, p = 0.4716. WT vs hiw p = 0.2168 (NS), WT vs hiw > dNmnat-RNAi p = 0.1882 (NS), hiw vs hiw > dNmnat-RNAi p = 0.9966 (NS)].(E) Representative electrophysiological traces from a synaptic facilitation experiment in which trains of 5 pulses were used to evoke consecutive action potentials at 100 ms ISI. Genotypes for these experiments were: WT (elav>UAS-RFP), hiw mutant (hiw; elav>UAS-RFP), or hiw depleted of dNmnat (hiw; elav>dNmnat-RNAi). (F) Quantification of the facilitation index, in which the amplitude of the 5th pulse is divided by the amplitude of the 1st pulse. [100 ms ISI: WT n=11, hiw n= 9, hiw>dNmnat-RNAi n= 12. 1-way ANOVA w/Tukey's multiple comparisons, DF = 31, F= 7.546, p =0.0023. WT vs hiw p =0.0020 (**), hiw vs hiw> dNmnat-RNAi p= 0.0229 (*), WT vs hiw> dNmnat-RNAi p= 0.5141 (NS). 50 ms ISI: WT n=11, hiw n= 9, hiw>dNmnat-RNAi n= 12. 1-way ANOVA w/Tukey's multiple comparisons, DF = 31, F= 5.587, p =0.0089. WT vs hiw p =0.0129 (*), hiw vs hiw> dNmnat-RNAi p= 0.0217 (*), WT vs hiw> dNmnat-RNAi p= 0.9546 (NS).]
Figure 4(A) Representative images of NMJ synaptic terminals in control (DVGlut-Gal4 > UAS-HA-dNmnat) and hiw mutant (hiwND9,DVGlut-Gal4 > UAS-HA-dNmnat) larvae. NMJs at larval muscle 4 were identified using HRP (blue in top panel) immunostaining. Insets highlight active zones within single boutons, which were identified by staining for the presynaptic active zone protein Brp (red). An anti-HA antibody was used to stain for an HA-tagged dNmnat (green) transgene to measure protein levels in control and hiw mutant active zones.(B) Quantification of HA-dNmnat levels across the entire NMJ synaptic terminal, normalized to total terminal area and presented as fold change over WT. [WT > HA-dNmnat n = 10 terminals, hiw > HA-dNmnat n = 10 terminals. Unpaired T-test, DF = 18, p = 0.0028 (**)].(C) Quantification of HA-dNmnat levels per unit area in WT and hiw for synaptic terminals excluding active zones (herein referred to as "terminals") versus active zones (AZs) only. [WT n = 10 terminals, hiw n = 9 terminals, each parsed into terminal minus AZs and AZs only. 2-way ANOVA with Tukey's multiple comparisons, DF = 34, p = 0.0005 for row factor (terminal versus AZs) and p < 0.0001 for column factor (WT vs hiw). WT terminal vs hiw terminal p = 0.9830 (NS). Hiw terminal vs hiw AZs p < 0.0001 (****). WT AZs vs hiw AZs p < 0.0001 (****).]
Figure 5A) Representative electron micrographs of entire boutons in wild type, hiw;wnd, and hiw;wnd, Elav-GAL4 > dNmnat RNAi NMJs.B) High magnification electron micrographs showing representative "T-bar" structures at active zones in NMJs of the indicated genotypes. Red arrows denote the beginning and end of the T-bar top, demonstrating changes in T-bar width in the genotypes tested. As illustrated, a significant reduction in T-bar size is observed in hiw;wnd, and this defect is suppressed in hiw;wnd, Elav-GAL4 > dNmnat RNAi.C) Quantification of mean T-bar width [WT n = 90, hiw;wnd n = 55, hiw;wnd, Elav-GAL4 > dNmnat RNAi n=81. 1-way ANOVA w/Tukey's multiple comparisons, DF=225, F=13.829, p < 0.0001. WT vs hiw;wnd p < 0.0001 (****), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p = 0.9318 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p < 0.0001 (****)]. (D) Analysis of cumulative frequency distribution of T-bar width shows a left shift in distribution in hiw;wnd but no change in hiw;wnd, Elav-GAL4 > dNmnat RNAi compared with WT NMJs [WT vs hiw;wnd K-S test, D=0.5636, p < 0.0001 (****), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.1815, p = 0.1240 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.4121, p < 0.0001 (****)].]. (E) Quantification of mean T-bar height [WT n = 90, hiw;wnd n = 55, hiw;wnd, Elav-GAL4 > dNmnat RNAi n=81. 1-way ANOVA w/Tukey's multiple comparisons, DF=225, F=10.472, p < 0.0001. WT vs hiw;wnd p = 0.0008 (***), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p = 0.6694 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p < 0.0001 (****)] (F) Analysis of cumulative frequency distribution of T-bar height shows a left shift in distribution in hiw;wnd but no change in hiw;wnd, Elav-GAL4 > dNmnat RNAi compared with WT NMJs [WT vs hiw;wnd K-S test, D=0.3343, p = 0.001 (***), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.1012, p = 0.7748 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.3484, p = 0.0008 (***)].
Figure 6(B) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), or one of the catalytic dNmnat mutants (DVGlut-Gal4 > UAS-WR-dNmnat, DVGlut-Gal4 > UAS-H30A-dNmnat).(C) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 8 cells, UAS-WR-dNmnat n = 7 cells, UAS-H30A-dNmnat n= 7 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=21, F=0.8596, p = 0.4383. UAS-RFP vs UAS-WR-dNmnat p = 0.8810 (NS), UAS-RFP vs UAS-H30A-dNmnat p = 0.9997 (NS)].(D) Quantification of quantal content (±SEM) [UAS-RFP n = 8 cells, UAS-WR-dNmnat n = 7 cells, UAS-H30A-dNmnat n= 7 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=21, F=1.154, p = 0.3365. UAS-RFP vs UAS-WR-dNmnat p = 0.8696 (NS), UAS-RFP vs UAS-H30A-dNmnat p = 0.9697 (NS)].]. (E) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing an RNAi to knockdown NAD+syn (hiwND8;Elav-Gal4 > UAS-NAD+syn-RNAi).(F) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > NADsyn-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=29, F=14.02, p<0.0001. WT vs hiw p <0.0001 (****), WT vs hiw > NADsyn-RNAi p = 0.5147 (NS)].]. (G) Quantification of quantal content. [WT n = 10 cells, hiw n = 10 cells, hiw > NADsyn-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=29, F= 6.635, p = 0.0047. WT vs hiw p = 0.0063 (**), WT vs hiw > NADsyn-RNAi p = 0.8942 (NS)].(H) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels E-G. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red).(I) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 25, hiw n = 21, hiw > NADsyn-RNAi n=24. 1-way ANOVA w/Tukey's multiple comparisons, DF=67, F=141.3, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Nadysn-RNAi p > 0.0001 (****)].
Figure 1Single plane confocal fluorescent microscopy images of 5-day-old animals harboring GFP-tagged endogenous HSP-110 (HSP-110::GFP). Muscle cells are outlined. M: muscle, H: hypodermis. In animals that co-express hairpin constructs targeting hsp-110 in muscle cells (HSP-110;HPI and HSP-110;HPII), GFP fluorescence is strongly reduced in muscle cells in comparison to control animals, indicating HSP-110::GFP depletion specifically in muscle cells. Scale bar: 10 µm.Experimental set-up: Age-synchronized 4-day-old animals were subjected to heat stress for 3 h at 33°C (+HS) then returned to 20°C or left at 20°C (-HS), respectively. Arrows indicate imaging time points. The -HS controls were imaged at the same time point as the +HS animals after 24h at 20°C. Maximum intensity projections of fluorescent microscopy z-stacks of animals expressing the indicated transgenes. HS-induced FLUCSM::GFP foci are cleared after a recovery for 24h at 20°C in the WT background, but persist in the presence of the hsp-110 hairpin construct. Scale bar: 20 µm. C Quantification of FLUCSM::GFP foci disaggregation. % disaggregation is calculated as 100 - ratio of FLUCSM::GFP foci area relative to total muscle area at the +HS 24h time point to the +HS 2h time point. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's adjustment for multiple comparisons. ** p ≤ 0.01. Data information: All strains harbor the sid-1(pk3321) allele.