Preadipocytes bearing shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine . expression was analyzed on day 6 . n = 3 biological replicates .

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Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine. Protein expression was analyzed on day 6. n=3 biological replicates.


Brown preadipocytes were infected by lentiviral shRNAs against Asns . were differentiated in culture media with or without 1mM asparagine . Western blot was performed on day 6 .

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Brown preadipocytes were infected by lentiviral shRNAs against Asns. Cells were differentiated in culture media with or without 1mM asparagine. Western blot was performed on day 6.


UCP1 PPARγ in BAT inguinal WAT ( iWAT ) from mice in ( E ) .

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UCP1 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 were subjected to indirect calorimetry at both basal ( I ) CL316 , 243 - stimulated ( J ) states . n = 5 mice each group . Data information : Data represent means ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - Student ' s t .

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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.


Rectal in mice of ( A ) during cold exposure . n = 5 mice each group .

Rectal temperature in mice of (A) during cold exposure. n=5 mice each group.


BAT inguinal WAT ( iWAT ) gene expression in mice of ( A ) .

BAT and inguinal WAT (iWAT) gene expression analysis in mice of (A).


Hematoxylin eosin of BAT , inguinal WAT ( iWAT ) epididymal WAT ( eWAT ) of mice in ( A ) .

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Hematoxylin and eosin staining of BAT, inguinal WAT (iWAT) and epididymal WAT (eWAT) of mice in (A).


Glucose tolerance was performed after a high - fat diet feeding for 17 in mice of ( A ) . n = 5 mice each group . Serum insulin in mice of ( A ) . n = 5 mice each group . Serum triglyceride in mice of ( A ) . n = 5 mice each group . Data information : Data represent means ± . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - Student ' s t .

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Glucose 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 inguinal WAT ( iWAT ) UCP1 PPARγ in mice of ( A ) .

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BAT and inguinal WAT (iWAT) UCP1 and PPARγ protein levels in mice of (A).


Heat map of metabolite relative in brown fat treated with or without 1mM asparagine .

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Heat map of metabolite relative levels in brown fat cells treated with or without 1mM asparagine.


Immunoblot detection of glucose transporters , glycolysis TCA key enzymes in brown fat Asparagine was supplemented to during differentiation .

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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 glucose transporters , glycolysis TCA key enzymes in immortalized brown fat ( E ) , BAT from mice fed asparagine ( F ) injected asparaginase ( G ) . Asparagine was supplemented to during differentiation .

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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.


Intracellular abundance of 13C - isotopologue metabolites downstream of glycolysis in brown adipocytes . n = 5 independent experiments . Data information : Data represent means ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - Student ' s t .

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Intracellular 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 . of glucose ( C ) lactate ( D ) from media . 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 ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - Student ' s t .

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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.


of intracellular acid in brown fat ( day 6 ) treated with or without 1mM asparagine . n = 4 independent experiments . Data information : Data represent means ± . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - Student ' s t .

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Quantification 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 phosphorylation of 4E - BP1 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 .

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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 for 48h . Then western blot detected expression on day 6 .

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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 . expression was performed on day 6 .

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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.


F . Dopaminergic neuronal cultures from control lines glucocerebrosidase ( GBA ) N370S PD were immunoblotted for presence of LAG3 . No band for LAG3 could be observed in neurons .

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F. 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 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 D2G40 ; left panel zoomed - in insets ) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 ( DOX ON ; right panel zoomed - in insets ) . Scale bars 25 µm .

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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 presence of LAG3 . No band for LAG3 could be evidenced in any of brain homogenates . Control samples ( tonsils activated human T ) show expected bands .

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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.


B . Mixed neuroglial , neuron - enriched , glial cortical cultures were assayed for presence of LAG3 where no band could be found . Activated T display band at expected .

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B. 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 scanning confocal microscopy , no mouse LAG3 signal could be detected in murine neuronal cultures ( Auto - mLAG3 transduced , DOX OFF non - transduced neurons ) using anti - mouse LAG3 antibody ( MABF954 ) whereas LAG3 was clearly detected in neurons induced to express mLAG3 ( Auto - mLAG3 transduced , DOX ON ) . Scale bars 20 µm .

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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 cDNA copy number . Mixed neuroglial cultures , glial cultures , neuron - enriched cultures show transcript that are close to indistinguishable to those from LAG3 ‑ / ‑ BH . Conversely , around 1600 transcripts / ng of were counted in activated T .

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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.


A . B . Binding of ASC filaments ( A ) or α - synuclein PFFs ( B ) to multiple . Serial dilutions of ASC filaments α - synuclein PFFs were performed bound fractions were detected using anti - ASC or anti - α - synuclein ( MJFR1 ) antibodies . ASC filaments did not interact with any of ( A ) . Conversely , α - synuclein PFFs strongly bound LAG3 , CD4 , MTBD tau showed moderate binding properties to PrPC , APOE3 , TDP - 43 . No binding between α - synuclein PFFs nAra h 2 or BSA could be detected . Values represent means ± SD of technical duplicates .

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A. 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 . D . Diffusional sizing of α - synuclein PFFs with fluorescently conjugated LAG3 , CD4 , BSA , MJFR1 α - synuclein antibody ( C ) of fluorescently labelled LAG3 with monomeric α - synuclein , α - synuclein PFFs , FGL1 , Relatlimab anti - LAG3 antibody ( D ) . An increase in hydrodynamic radius ( Rh ) of α - synuclein PFFs is seen with increasing concentrations for MJFR1 but not for LAG3 or any of control , also not at a lower scale ( see insert ) ( C ) . Similarly , hydrodynamic radius of labelled LAG3 increases with higher concentrations of Relatlimab FGL1 but does not change with α - synuclein PFFs or monomeric α - synuclein ( D ) . Values are given as means ± SD of technical triplicates .

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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 . F . Co - immunoprecipitation of LAG3 subsequent immunoblotting with anti α - synuclein antibody ( MJFR1 ) ( E ) co - immunoprecipitation of α - synuclein ( monomeric or PFFs ) subsequent immunoblotting with anti - LAG3 antibody ( D2G40 ) ( F ) . Using anti - α - synuclein antibody for pulldown resulted in LAG3 bands with both monomeric as well as fibrillar α - synuclein . usage of anti - LAG3 for pulldown led to specific detection of α - synuclein PFFs , although PFF fraction predominates also in input supernatant . Bands for α - synuclein ( E ) LAG3 ( F ) are indicated by arrows .

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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.


Human neural cultures transduced by Auto - hLAG3 were treated by α - synuclein PFFs 4 days post LAG3 expression induction by DOX kept in culture for 2 or 4 . Both transgenic ( hLAG3 D2G40 - positive in ( A ) 17B4 - positive in ( B ) ) non - transduced , wild - type neurons ( selected neurons in zoomed - in insets ) propagated α - synuclein developed characteristic pS129 - positive ( 81A - positive in ( A ) EP1536Y - positive in ( B ) ) α - synuclein . Scale bars 25 µm . Trained ilastik algorithms were used to segment pixels of 81A , EP1536Y MAP2 stainings imaged by high - content wide - field microscope , which were used to quantify signal of 81A - positive ( C ) EP1536Y - positive ( D ) α - synuclein expressed as % of MAP2 - positive . Almost entire wells ( 182 fields per well ) were imaged for every condition replicate each datapoint in plot represents 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 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 p = 0 . 2522 at 4 ; p = 0 . 9986 for EP1536Y at 2 p = 0 . 3042 at 4 ) .

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Human 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).


A . 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 α - synuclein TG LAG3 + / + at 268 d . survivals of ASYNA53T LAG3 ‑ / ‑ , LAG3 + / - LAG3 + / + mice were similar ( Mantel - Cox log - rank , p - value = 0 . 165 ) . Mice were euthanized dissected in late - stage symptomatic phase , which would inevitably result in within a week . None of control LAG3 ‑ / ‑ α - synuclein WT mice died over course of experiment .

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A. 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 ( pS129 ) aggregated ( Thioflavin S ) in sections of end - stage symptomatic mice ; no obvious difference in terms of pattern or number of stained were apparent in midbrain or brain . Scale bars represent 1 mm ( sagittal section ) 100 µm ( panel ) .

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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 of pS129 - positive α - synuclein inclusions in hippocampal slice cultures ( HSCs ) at 5 post - seeding with 350 µM PFF . Scale bars represent 500 µm ( overview ) , 100 µm ( insets ) . Shown is mean ± 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 p > 0 . 9808 for LAG3 WT vs . KO in A53T WT .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

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.


( A ) Representative EPSP 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O

(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 ) of mean ( ± ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per , then amplitudes were averaged per genotype . [ UAS - RFP n = 13 , UAS - dNmnat n = 14 , UAS - Wnd n = 10 . 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 ( * * ) ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(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 ) of mean ( ± ) mEPSP amplitudes , in which 75 consecutive spontaneous events were averaged per , then amplitudes were averaged per genotype . [ UAS - RFP n = 15 , UAS - dNmnat n = 14 , UAS - Wnd n = 10 . 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 ( * * * ) ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

]. (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 ) of quantal content , which was calculated individually per by dividing mean EPSP amplitude by mean mEPSP amplitude , then averaged per genotype . [ UAS - RFP n = 15 , UAS - dNmnat n = 14 , UAS - Wnd n = 10 . 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 ( * * * ) ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(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 of NMJ synaptic terminal at muscle 4 in third - instar larvae . genotypes in this experiment were identical to those tested in electrophysiology experiments in A - D . NMJs were stained for presynaptic bouton marker DVGLUT ( green ) nerve marker HRP ( red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

]. (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 ) of mean ( ± ) 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 ( * * * * ) ] .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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(****)].


( A ) Representative EPSP 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 ) , hiw mutants expressing RNAis to knockdown either dNmnat ( hiwND8 ; Elav - Gal4 > UAS - dNmnat - RNAi [ # 29402 ] ) or Wnd ( hiwND8 ; Elav - Gal4 > UAS - Wnd - RNAi [ # 25396 ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(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 ) of mean ( ± ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per , then amplitudes were averaged per genotype . [ WT n = 10 , hiw n = 10 , hiw > Wnd - RNAi n = 10 , hiw > dNmnat - RNAi n = 10 . 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 ) ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(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 ) of quantal content ( ± ) . [ WT n = 9 , hiw n = 9 , hiw > Wnd - RNAi n = 10 , hiw > dNmnat - RNAi n = 10 . 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 ) ] .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(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 of NMJ synaptic terminal at muscle 4 in third - instar larvae . genotypes in this experiment were identical to those tested in panels A - C . NMJs were stained for presynaptic bouton marker DVGLUT ( green ) nerve marker HRP ( red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

(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 ) of mean ( ± ) 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 ) ] .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(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)].


( A ) Representative of immunostained larval NMJs active zones from WT , hiw mutant , hiw mutant driving an RNAi against dNmnat [ # 29402 ] . In top 3 panels , greyscale of entire terminal were acquired by for nerve marker HRP . Presynaptic Brp ( green ) postsynaptic dGluRIII clusters ( magenta ) were imaged to quantify N in each genotype .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O

(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 ) of NMJ synaptic terminal surface 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 ) ] .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(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 ) of BRP 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 ) ] .

O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(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 ) of of Brp 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 ) ] .

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(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 ) of facilitation index , in which amplitude of 5th is divided by amplitude of 1st . [ 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 ) . ]

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(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).]


( A ) Representative of NMJ synaptic terminals in control ( DVGlut - Gal4 > UAS - HA - dNmnat ) 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 for presynaptic active zone Brp ( red ) . An anti - HA antibody was used to stain for an HA - tagged dNmnat ( green ) transgene to measure in control hiw mutant active zones .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O

(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 ) of HA - dNmnat across entire NMJ synaptic terminal , normalized to total terminal presented as fold change over WT . [ WT > HA - dNmnat n = 10 terminals , hiw > HA - dNmnat n = 10 terminals . Unpaired T - , DF = 18 , p = 0 . 0028 ( * * ) ] .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) of HA - dNmnat per unit in WT 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 AZs only . 2 - way ANOVA with Tukey ' s multiple comparisons , DF = 34 , p = 0 . 0005 for row factor ( terminal versus AZs ) 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 ( * * * * ) . ]

O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(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 (****).]


A ) Representative electron of entire boutons in wild type , hiw ; wnd , hiw ; wnd , Elav - GAL4 > dNmnat RNAi NMJs .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O

A) Representative electron micrographs of entire boutons in wild type, hiw;wnd, and hiw;wnd, Elav-GAL4 > dNmnat RNAi NMJs.


B ) High magnification electron showing representative " T - bar " structures at active zones in NMJs of indicated genotypes . Red arrows denote beginning end of T - bar top , demonstrating changes in T - bar width in genotypes tested . As illustrated , a significant reduction in T - bar is observed in hiw ; wnd , this defect is suppressed in hiw ; wnd , Elav - GAL4 > dNmnat RNAi .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O

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 ) 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 ) 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 , D = 0 . 5636 , p < 0 . 0001 ( * * * * ) , WT vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi K - S , D = 0 . 1815 , p = 0 . 1240 ( NS ) , hiw ; wnd vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi K - S , D = 0 . 4121 , p < 0 . 0001 ( * * * * ) ] .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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 ) 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 ) 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 , D = 0 . 3343 , p = 0 . 001 ( * * * ) , WT vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi K - S , D = 0 . 1012 , p = 0 . 7748 ( NS ) , hiw ; wnd vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi K - S , D = 0 . 3484 , p = 0 . 0008 ( * * * ) ] .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

]. (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 (***)].


( B ) Representative EPSP 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 catalytic dNmnat mutants ( DVGlut - Gal4 > UAS - WR - dNmnat , DVGlut - Gal4 > UAS - H30A - dNmnat ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O

(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 ) of mean ( ± ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per , then amplitudes were averaged per genotype . [ UAS - RFP n = 8 , UAS - WR - dNmnat n = 7 , UAS - H30A - dNmnat n = 7 . 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 ) ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(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 ) of quantal content ( ± ) [ UAS - RFP n = 8 , UAS - WR - dNmnat n = 7 , UAS - H30A - dNmnat n = 7 . 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 ) ] .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(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 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 ) , hiw mutants expressing an RNAi to knockdown NAD + syn ( hiwND8 ; Elav - Gal4 > UAS - NAD + syn - RNAi ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

]. (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 ) of mean ( ± ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per , then amplitudes were averaged per genotype . [ WT n = 10 , hiw n = 10 , hiw > NADsyn - RNAi n = 10 . 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 ) ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

(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 ) of quantal content . [ WT n = 10 , hiw n = 10 , hiw > NADsyn - RNAi n = 10 . 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 ) ] .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

]. (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 of NMJ synaptic terminal at muscle 4 in third - instar larvae . genotypes in this experiment were identical to those tested in panels E - G . NMJs were stained for presynaptic bouton marker DVGLUT ( green ) nerve marker HRP ( red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

(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 ) of mean ( ± ) 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 ( * * * * ) ] .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(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 (****)].


Single plane confocal microscopy of 5 - day - old harboring GFP - tagged endogenous HSP - 110 ( HSP - 110 : : GFP ) . Muscle are outlined . M : muscle , H : hypodermis . In that co - express hairpin constructs targeting hsp - 110 in muscle ( HSP - 110 ; HPI HSP - 110 ; HPII ) , GFP is strongly reduced in muscle in comparison to control , indicating HSP - 110 : : GFP depletion specifically in muscle . Scale bar : 10 µm .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O

Single 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 : - synchronized 4 - day - old 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 . - HS controls were imaged at same time point as + HS after 24h at 20 ° C . Maximum projections of microscopy z - stacks of expressing indicated transgenes . HS - induced FLUCSM : : GFP foci are cleared after a recovery for 24h at 20 ° C in WT background , but persist in presence of hsp - 110 hairpin construct . Scale bar : 20 µm . C of FLUCSM : : GFP foci disaggregation . % disaggregation is calculated as 100 - of FLUCSM : : GFP foci relative to total muscle at + HS 24h time point to + HS 2h time point . Data are displayed as mean ± ( in % ) ( N = 3 ) . Statistical was done using one - way ANOVA with Dunnett ' s adjustment for multiple comparisons . * * p ≤ 0 . 01 . Data information : strains harbor sid - 1 ( pk3321 ) allele .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A Maximum projections of microscopy z - stacks of 5 - day - old nematodes expressing indicated transgenes are shown . dashed lines outline borders of muscle . Scale bar : 10 µm . co - expression of HPI HPII reduces amount of α - Syn : : YFP inclusions . B of α - Syn : : YFP foci in 3 - to 6 - day - old . Product of mean foci foci relative to muscle is displayed ( relFluoFoci ) . Data are shown as mean ± ( in % ) ( N = 3 ) . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . * * = p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Maximum intensity projections of fluorescent microscopy z-stacks of 5-day-old nematodes expressing the indicated transgenes are shown. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. The co-expression of HPI and HPII reduces the amount of α-Syn::YFP inclusions. B Quantification of α-Syn::YFP foci in 3- to 6-day-old animals. Product of mean foci fluorescence and foci area relative to muscle area is displayed (relFluoFoci). Data are shown as mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. ** = p ≤ 0.01, *** p ≤ 0.001.


C Motility as a measure for transgene toxicity . Displayed is mean number of bends per 30 s ± ( N = 3 ) . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Motility Assay as a measure for transgene toxicity. Displayed is the mean number of body bends per 30 s ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. ** p ≤ 0.01, *** p ≤ 0.001.


D Maximum projections of microscopy z - stacks of 6 - day - old expressing indicated transgenes . Scale bar : 100 µm . co - expression of HPI HPII reduces amount of Q35 : : YFP inclusions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Maximum intensity projections of fluorescent microscopy z-stacks of 6-day-old animals expressing the indicated transgenes. Scale bar: 100 µm. The co-expression of HPI and HPII reduces the amount of Q35::YFP inclusions.


E of Q35 : : YFP foci in WT hsp - 110 hairpin background . Shown is mean ± ( N = 3 ) . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Quantification of Q35::YFP foci in WT and hsp-110 hairpin background. Shown is the mean ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01.


F Motility as a measure for transgene toxicity . Displayed is mean number of bends per 30 s ± ( N = 3 ) . Statistical was done using one - way ANOVA with Dunnett ' s multiple comparisons . * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Motility Assay as a measure for transgene toxicity. Displayed is the mean number of body bends per 30 s ± SEM (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01.


G Maximum projections of microscopy z - stacks of of 5 - day - old nematodes expressing indicated transgenes . dashed lines outline borders of muscle . Scale bar : 10 µm . M : muscle , H : hypodermis . Signal outside of muscle reveals spreading of α - Syn . H of showing α - Syn transmission at indicated ages . Displayed is mean ± ( in % ) ( N = 4 ) . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . * * * p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Maximum intensity projections of fluorescent microscopy z-stacks of of 5-day-old nematodes expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. M: muscle, H: hypodermis. Signal outside of muscle cells reveals spreading of α-Syn. H Quantification of animals showing α-Syn transmission at indicated ages. Displayed is the mean ± SEM (in %) (N = 4). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. *** p ≤ 0.001.


A Maximum projections of confocal microscopy z - stacks of 5 - day - old expressing indicated transgenes . dashed lines outline borders of muscle . Scale bar : 10 µm . expression of yeast SSE1N575Y E575A mutant cannot reverse reduction of α - Syn : : YFP foci in hsp - 110 hairpin expressing .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

A Maximum intensity projections of confocal microscopy z-stacks of 5-day-old animals expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. The expression of the yeast SSE1N575Y E575A mutant cannot reverse the reduction of α-Syn::YFP foci in the hsp-110 hairpin expressing animals.


B of motility as a measure for transgene toxicity . Displayed is mean ± ( N = 3 ) amount of bends / 30s of expressing indicated transgenes . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . n . s . = not significant , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . presence of yeast SSE1N575Y E575A mutant was not able to suppress rescue of α - Syn : : YFP toxicity in hsp - 110 hairpin expressing .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

B Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes. Statistical analysis was done using two-way ANOVA and with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001. The presence of the yeast SSE1N575Y E575A mutant was not able to suppress the rescue of α-Syn::YFP toxicity in hsp-110 hairpin expressing animals.


C Maximum projections of microscopy z - stacks of 5 - day - old nematodes expressing indicated transgenes . dashed lines outline borders of muscle . M : muscle , H : hypodermis . Scale bars : 10 µm . Signal outside of muscle reveals spreading of α - Syn .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

C Maximum intensity projections of fluorescent microscopy z-stacks of 5-day-old nematodes expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. M: muscle, H: hypodermis. Scale bars: 10 µm. Signal outside of muscle cells reveals spreading of α-Syn.


D of showing α - Syn transmission into hypodermis at indicated ages . Displayed is mean ± ( in % ) ( N = 3 ) . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . n . s . = not significant , * * * p ≤ 0 . 001 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Quantification of animals showing α-Syn transmission into the hypodermis at indicated ages. Displayed is the mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, *** p ≤ 0.001.


E Experimental set - up : - synchronized 4 - day - old 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 . - HS controls were imaged at same time point as + HS after 24h at 20 ° C . Maximum projections of microscopy z - stacks of expressing indicated transgenes . Scale bar : 20 µm . impaired clearance of HS - induced FLUCSM : : GFP foci in hsp - 110 hairpin expressing is rescued by co - expression of WT but not by HSP - 70 binding deficient SSE1N575Y E575A mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

E 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. Scale bar: 20 µm. The impaired clearance of HS-induced FLUCSM::GFP foci in the hsp-110 hairpin expressing cells is rescued by co-expression of WT but not by the HSP-70 binding deficient SSE1N575Y E575A mutant.


F of FLUCSM : : GFP foci disaggregation . % disaggregation is calculated as 100 - of FLUCSM : : GFP foci relative to total muscle at + HS 24h time point to + HS 2h time point . Data are displayed as mean ± ( in % ) ( N = 3 ) . Statistical was done using one - way ANOVA with Dunnett ' s multiple comparison . n . s . = not significant , * * p ≤ 0 . 01 . strains harbor sid - 1 ( pk3321 ) allele .

O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F 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 multiple comparison test. n.s. = not significant, ** p ≤ 0.01. All strains harbor the sid-1(pk3321) allele.


A Maximum projections of confocal z - stacks of 4 - day - old expressing indicated transgenes or harboring indicated - sensitive ( ts ) mutations . Muscle were stained with anti - myosin ( green ) Alexa Flour 647 - phalloidin ( purple ) . Scale bar : 5 µm . Myosin misfolding reflects exposure of ts mutant phenotype of myosin ( ts ) [ unc - 54 ( e1301 ) ] at permissive temperate of 15 ° C by co - expression of hsp - 110 hairpins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O

A Maximum intensity projections of confocal z-stacks of 4-day-old animals expressing the indicated transgenes or harboring the indicated temperature-sensitive (ts) mutations. Muscle cells were stained with anti-myosin (green) and Alexa Flour 647-phalloidin (purple). Scale bar: 5 µm. Myosin misfolding reflects the exposure of the ts mutant phenotype of myosin(ts) [unc-54(e1301)] at the permissive temperate of 15°C by co-expression of the hsp-110 hairpins.


B of motility as a measure for toxicity . Displayed is mean ± ( N = 4 ) of bends / 30s of expressing indicated transgenes or harboring indicated mutations . Statistical was done using one - way ANOVA with Dunnett ' s multiple comparison . * * * p ≤ 0 . 001 . Co - expression of hsp - 110 hairpins exposed ts mutant phenotype at permissive temperate of 15 ° C , resulting in a significant increase in movement defects . strains contain sid - 1 ( pk3321 ) allele .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Quantification of motility as a measure for toxicity. Displayed is the mean ± SEM (N = 4) of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparison test. *** p ≤ 0.001. Co-expression of the hsp-110 hairpins exposed the ts mutant phenotype at the permissive temperate of 15°C, resulting in a significant increase in movement defects. All strains contain the sid-1(pk3321) allele.


C of motility as a measure for transgene toxicity . Displayed is mean ± ( N = 4 ) amount of bends / 30s of expressing indicated transgenes or harboring indicated mutations . Statistical was done using two - way ANOVA with Tukey ' s multiple comparison . * p ≤ 0 . 05 . Expression of hsp - 110 hairpins caused movement defects with increasing .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O

C Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 4) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Tukey's multiple comparison test. * p ≤ 0.05. Expression of the hsp-110 hairpins caused movement defects with increasing age.


D Maximum projections of confocal z - stacks of 12 - day - old expressing indicated transgenes . Scale bar : 10 µm . FLUCSM forms foci with increasing , which is markedly accelerated upon hsp - 110 KD .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O

D Maximum intensity projections of confocal z-stacks of 12-day-old animals expressing the indicated transgenes. Scale bar: 10 µm. FLUCSM forms foci with increasing age, which is markedly accelerated upon hsp-110 KD.


E of with FLUCSM : : GFP foci at indicated days of life . Data are displayed as mean ± ( in % ) ( N = 3 ) . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . n . s . = not significant , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Quantification of animals with FLUCSM::GFP foci at indicated days of life. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001.


F of motility as a measure for transgene toxicity . Displayed is mean ± ( N = 3 ) amount of bends / 30s of expressing indicated transgenes or harboring indicated mutations . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . n . s . = not significant , * * * p ≤ 0 . 001 . Expression of hsp - 110 hairpins caused movement defects with increasing . beneficial effects of HSP - 110 KD on amyloid toxicity is masked in older .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

F Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, *** p ≤ 0.001. Expression of the hsp-110 hairpins caused movement defects with increasing age. The beneficial effects of HSP-110 KD on amyloid toxicity is masked in older animals.


G of motility as a measure for transgene toxicity . Displayed is mean ± ( N = 3 ) amount of bends / 30s of expressing indicated transgenes or harboring indicated mutations . Statistical was done using two - way ANOVA with Dunnett ' s multiple comparison . n . s . = not significant , , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . Expression of hsp - 110 hairpins caused movement defects with increasing . beneficial effects of HSP - 110 KD on amyloid toxicity is masked in older . Data information : strains harbor sid - 1 ( pk3321 ) allele .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

G Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, , ** p ≤ 0.01, *** p ≤ 0.001. Expression of the hsp-110 hairpins caused movement defects with increasing age. The beneficial effects of HSP-110 KD on amyloid toxicity is masked in older animals. Data information: All strains harbor the sid-1(pk3321) allele.


A ) Left : Representative current traces from wildtype NavMs channels showing first 0 . 1 s of 0 . 5 s activations from - 180 mV holding potential recorded with indicated extracellular cations . Right : Resulting current - voltage profiles profiles , where current magnitudes were normalized to maximum inward current measured in sodium condition . B ) Left : Representative current traces from mutant channel E178D ( as in A ) . Right : Resulting current - voltage profiles ( error ± ; n = 6 - 9 for NavMs ; n = 4 - 8 for E178D ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

A) Left: Representative current traces from wildtype NavMs channels showing the first 0.1 s of 0.5 s activations from -180 mV holding potential recorded with the indicated extracellular cations. Right: Resulting current-voltage profiles profiles, where current magnitudes were normalized to the maximum inward current measured in the sodium condition.B) Left: Representative current traces from mutant channel E178D (as in A). Right: Resulting current-voltage profiles (error ± SEM; n = 6-9 for NavMs; n = 4-8 for E178D).


A ) Upper : Overview of AB tetramer , with each of monomers depicted in a different colour . front monomer has been removed for clarity . black box indicates region expanded in lower panel . Lower : An expanded view of selectivity filter , showing only two monomers as pink ribbon cartoons with selectivity filter residues drawn as sticks in corresponding colours . Sodium ions are shown as cyan spheres well - ordered waters as red spheres ( at 0 . 4x their actual radii , for clarity ) . final refined 2Fo - Fc electron density maps for , ions water are shown at 2 . 0 σ in grey mesh 0 . 8 σ in grey , initial difference map ( without sodiums or waters included in phase calculation ) at 3 . 0 σ in green mesh . B ) Superposition of sodium ion binding sites found in crystallographically - distinct AB CD tetramers proposed binding sites from other studies . tetramers were superposed with LSQKAB program ( Kabsch , 2010 ) from CCP4 ( Winn et al . , 2011 ) using residues 131 - 220 from four monomers in each tetramer . Only two monomers from AB tetramer are shown for clarity , coloured as in panel A . Sodium ions from AB tetramer are depicted as cyan spheres , those from CD tetramer as teal spheres . Predicted sodium ion binding sites from Ulmschneider et al . ( 2013 ) are indicated as small black spheres labelled on right of spheres according to nomenclature ( S0 to S4 ) in that study . Predicted sodium ion binding sites from Payendeh et al . ( 2011 ) , on a closed channel structure of close orthologue NavAb , are indicated on far right labelled as per nomenclature of that study ( SiteHFS , SiteCEN SiteIN ) . This subsequent molecular figures were drawn with PyMol ( PyMOL Molecular Graphics System , Version 1 . 6 , Schrödinger , LLC ) unless otherwise stated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Upper: Overview of the AB tetramer, with each of the monomers depicted in a different colour. The front monomer has been removed for clarity. The black box indicates the region expanded in the lower panel. Lower: An expanded view of the selectivity filter, showing only two monomers as pink and white ribbon cartoons with the selectivity filter residues drawn as sticks in the corresponding colours. Sodium ions are shown as cyan spheres and well-ordered waters as red spheres (at 0.4x their actual radii, for clarity). The final refined 2Fo-Fc electron density maps for the protein, ions and water are shown at 2.0 σ in dark grey mesh and 0.8 σ in light grey , and the initial difference map (without sodiums or waters included in the phase calculation) at 3.0 σ in green mesh.B) Superposition of the sodium ion binding sites found in the crystallographically-distinct AB and CD tetramers and the proposed binding sites from other studies. The tetramers were superposed with the LSQKAB program (Kabsch, 2010) from CCP4 (Winn et al., 2011) using residues 131-220 from all four monomers in each tetramer. Only two monomers from the AB tetramer are shown for clarity, and coloured as in panel A. Sodium ions from the AB tetramer are depicted as cyan spheres, those from the CD tetramer as teal spheres. Predicted sodium ion binding sites from Ulmschneider et al. (2013) are indicated as small black spheres and labelled on the right of the spheres according to the nomenclature (S0 to S4) in that study. Predicted sodium ion binding sites from Payendeh et al. (2011), based on a closed channel structure of the close orthologue NavAb, are indicated on the far right and labelled as per the nomenclature of that study (SiteHFS, SiteCEN and SiteIN). This and subsequent molecular figures were drawn with PyMol (The PyMOL Molecular Graphics System, Version 1.6, Schrödinger, LLC) unless otherwise stated.


A ) Top : Crystal structure overview of wildtype AB tetramer ( PDBID 5ZBZ ) , view as in Figure 2A . Left : Difference ( Fo - Fc ) electron density map ( mutant minus wildtype ) contoured at - 2 . 5σ drawn as red mesh , plotted on structure of wildtype ( E178 ) , highlighting consequences of loss of δ - carbon atom from residue 178 in E178D mutant . For reference , locations of sodium ion sites present in wildtype ( E178 ) structure are depicted as cyan spheres . Both structure ions are displayed as faded backgrounds so that difference map can be seen more clearly . Right : Final refined 2Fo - Fc electron density map for E178D mutant ( PDBID 4X88 ) , contoured at 1 . 5σ shown as grey mesh , with sodium ions depicted as cyan spheres . B ) Surface representations of ( left ) wildtype NavMs pore ( right ) E178D mutant coloured by electrostatic charge calculated by APBS ( Callenberg et al , 2010 ) drawn with VMD ( Humphrey et al . , 1996 ) , showing a slice through middle of tetramer . Sodium ions are shown as cyan spheres ; only 2 are present in mutant , instead of 3 seen in wildtype , with top sodium missing in mutant . lower panels are expanded views of selectivity filter regions , with E178 side chains depicted in yellow sticks location of main chain nitrogen of residue 179 indicated by black circle . alteration in structure caused by removal of methylene group of E178 exposes a positive region of polypeptide backbone adjacent to site where top sodium ion is located in wildtype ; this likely contributes to why an equivalent sodium ion is not seen adjacent to this position in mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Top: Crystal structure overview of the wildtype AB tetramer (PDBID 5ZBZ), view as in Figure 2A.Left: Difference (Fo-Fc) electron density map (mutant minus wildtype) contoured at -2.5σ and drawn as red mesh, plotted on the structure of the wildtype (E178) protein, highlighting the consequences of the loss of the δ-carbon atom from residue 178 in the E178D mutant. For reference, the locations of the sodium ion sites present in the wildtype (E178) structure are depicted as cyan spheres. Both the structure and the ions are displayed as faded backgrounds so that the difference map can be seen more clearly.Right: Final refined 2Fo-Fc electron density map for the E178D mutant (PDBID 4X88), contoured at 1.5σ and shown as grey mesh, with the sodium ions depicted as cyan spheres.B) Surface representations of (left) wildtype NavMs pore and (right) the E178D mutant coloured by electrostatic charge calculated by APBS (Callenberg et al, 2010) and drawn with VMD (Humphrey et al., 1996), showing a slice through the middle of the tetramer. Sodium ions are shown as cyan spheres; only 2 are present in the mutant, instead of the 3 seen in the wildtype, with the top sodium missing in the mutant. The lower panels are expanded views of the selectivity filter regions, with the E178 side chains depicted in yellow sticks and the location of the main chain nitrogen of residue 179 indicated by the black circle. The alteration in the protein structure caused by removal of the methylene group of E178 exposes a positive region of the polypeptide backbone adjacent to the site where the top sodium ion is located in the wildtype protein; this likely contributes to why an equivalent sodium ion is not seen adjacent to this position in the mutant.


Left : Sodium ions ( cyan ) in selectivity filter of NavMs sodium channel . Only two monomers ( in pink ) are shown for clarity . Coordinates are from AB tetramer of PDBID 5BZB . Right : Potassium ions ( blue ) in selectivity filter of KcsA potassium channel . As for NavMs structure , only two monomers ( in grey maroon ) are shown for clarity . Coordinates are from PDBID 1K4C ( Zhou et al . , 2004 ) . two crystal structures were first superimposed using SSM ( Krissinel Henrick , 2004 ) , are drawn using same scale , with correct between diameters of ions used , so that differences in internal dimensions of their selectivity filters can be seen , thus making it clear how much more space there is available in NavMs SF for intervening water molecules . In KcsA channel , ions are directly coordinated to polypeptide backbone atoms , whereas in NavMs SF , closest atoms ( side chains ) are obviously at longer distances than direct bonding to ions , enabling interactions via water molecules .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Left: Sodium ions (cyan) in the selectivity filter of the NavMs sodium channel. Only two monomers (in white and pink) are shown for clarity. Coordinates are from the AB tetramer of PDBID 5BZB.Right: Potassium ions (blue) in the selectivity filter of the KcsA potassium channel. As for the NavMs structure, only two monomers (in grey and maroon) are shown for clarity. Coordinates are from PDBID 1K4C (Zhou et al., 2004).The two crystal structures were first superimposed using SSM (Krissinel and Henrick, 2004), and are drawn using the same scale, and with the correct ratio between the diameters of the ions used, so that the differences in the internal dimensions of their selectivity filters can be seen, thus making it clear how much more space there is available in the NavMs SF for intervening water molecules. In the KcsA channel, the ions are directly coordinated to the polypeptide backbone atoms, whereas in the NavMs SF, the closest atoms (side chains) are obviously at longer distances than direct bonding to the ions, enabling interactions via water molecules.


C Upper panel : senescence curve of a representative est2 ∆ clone . samples were prepared at indicated time points to analyze telomere ( middle panel ) t - circles production ( lower panel graph ) during senescence immortalization . Telomeres were analyzed by Southern blot of XhoI cut hybridized with a TG1 - 3 probe . presence of t - circles was monitored by RCA analyzed by TG1 - 3 probed dot blot . graph shows signal intensities of dots measured with Fiji software ( arbitrary units ) . Similar results were obtained with at least 16 other est2 ∆ clones .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Upper panel: senescence curve of a representative est2∆ clone. DNA samples were prepared at the indicated time points to analyze telomere length (middle panel) and t-circles production (lower panel and graph) during senescence and cell immortalization. Telomeres were analyzed by Southern blot of the XhoI cut DNA hybridized with a TG1-3 probe. The presence of t-circles was monitored by RCA and analyzed by TG1-3 probed dot blot. The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units). Similar results were obtained with at least 16 other est2∆ clones.


E ) Dot blot of RCA product either treated or not with Exonuclease VII that degrades linear single - stranded in both 3 ' to 5 ' 5 ' to 3 ' directions ( upper panel ) . was hybridized with a telomeric TG1 - 3 probe . signal intensities of dots are shown ( lower graph ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Dot blot of the RCA product either treated or not with the Exonuclease VII that degrades linear single-stranded DNA in both the 3' to 5' and 5' to 3' directions (upper panel). The membrane was hybridized with a telomeric TG1-3 probe. The signal intensities of the dots are shown (lower graph).


A Senescence curve of a representative est2 ∆ clone corresponding dot blot of RCA product hybridized with indicated subtelomeric or telomeric probes . t - circle amounts were evaluated on signal of dots were expressed as fold increase over signal in absence of Φ29 ( lower graph ) .

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A Senescence curve of a representative est2∆ clone and corresponding dot blot of the RCA product hybridized with the indicated subtelomeric or telomeric probes. t-circle amounts were evaluated based on signal intensity of the dots and were expressed as fold increase over the signal in the absence of Φ29 (lower graph).


B Senescence curve of a representative est2 ∆ clone ( upper panel ) . samples were prepared at indicated time points to analyze telomere by Southern blot ( middle panel ) t - circles production by RCA performed in absence of dCTP dATP . ( lower panel ) . graph shows signal intensities of dots measured with Fiji software ( arbitrary units ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Senescence curve of a representative est2∆ clone (upper panel). DNA samples were prepared at the indicated time points to analyze telomere length by Southern blot (middle panel) and t-circles production by RCA assay performed in the absence of dCTP and dATP.(lower panel). The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units).