Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors . Control human R00 - 082 chondrocytes were serum starved in the presence of a GRK2 inhibitor , either CMPD101 ( C ) or paroxetine ( P ) , for 24 hours before they were treated with SAG for additional 24 hours ( C D ) . ( C ) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation ; actin levels served as a loading control . GLI3FL and GLI3R , full - length and repressor GLI3 variants , respectively . ( D ) Quantification of GLI3 and GLI1 levels by densitometry . Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine . Mean ± SEM . Mann - Whitney U test ( GLI3 ratio ) and Welch ´ s t - test ( GLI1 levels ) ; number of biological experiments is indicated .

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Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 24 hours (C D). (C) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation; actin levels served as a loading control. GLI3FL and GLI3R, full-length and repressor GLI3 variants, respectively. (D) Quantification of GLI3 and GLI1 levels by densitometry. Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine. Mean ± SEM. Mann-Whitney U test (GLI3 ratio) and Welch´s t-test (GLI1 levels); number of biological experiments is indicated.


Defective SMO cilia accumulation in chondrocytes with downregulated GRK2 . ( E ) Doxycycline ( DOX ) - inducible GRK2 downregulation in control human R00 - 082 chondrocytes , tested by western blot , normalized to actin and plotted below . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated . shScr , scramble shRNA .

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Defective SMO cilia accumulation in chondrocytes with downregulated GRK2. (E) Doxycycline (DOX)-inducible GRK2 downregulation in control human R00-082 chondrocytes, tested by western blot, normalized to actin and plotted below. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated. shScr, scramble shRNA.


( F ) Cells pre - treated with DOX for three days were serum starved , SAG treated , stained and analyzed as in ( A , B ) , and the intensity of ciliary SMO was analyzed and plotted . Note the impaired SMO translocation to cilia caused by GRK2 loss . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cilia are indicated . Scale bar , 1 µm .

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(F) Cells pre-treated with DOX for three days were serum starved, SAG treated, stained and analyzed as in (A,B), and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO translocation to cilia caused by GRK2 loss. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.


( A ) Control and GRK2 - / - fibroblasts were transfected with the TOPflash Firefly ( F ) luciferase vector together with a control Renilla ( R ) luciferase vector , and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual - luciferase assay . Note the impaired response to Wnt3A in GRK2 - / - cells . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated .

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(A) Control and GRK2-/- fibroblasts were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual-luciferase assay. Note the impaired response to Wnt3A in GRK2-/- cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.


( B Low phosphorylation ( p ) of Wnt3A co - receptor LRP6 as determined by western blot with pS1490 - and pT1572 - LRP6 specific antibodies , reduced LRP6 and DVL2 expression , and increased steady state phosphorylation of DVL2 . A representative experiment of four is shown . # , nonspecific band .

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(B Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. A representative experiment of four is shown. #, nonspecific band.


C ) Low phosphorylation ( p ) of Wnt3A co - receptor LRP6 as determined by western blot with pS1490 - and pT1572 - LRP6 specific antibodies , reduced LRP6 and DVL2 expression , and increased steady state phosphorylation of DVL2 . ( C ) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2 - / - cells , lower response of GRK2 - / - cells to Wnt3A by LRP6 phosphorylation at S1490 , and tendency towards the similar defect at T1572 . Densitometry also showed less of total DVL2 levels , and increased steady state pDVL2 in GRK2 - / - cells . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated .

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C) Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. (C) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2-/- cells, lower response of GRK2-/- cells to Wnt3A by LRP6 phosphorylation at S1490, and tendency towards the similar defect at T1572. Densitometry also showed less of total DVL2 levels, and increased steady state pDVL2 in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


( D ) Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in GRK2 - / - fibroblasts . Proximity ligation assay ( PLA ) between expressed FZD4 - GFP and ARRB2 - Flag showed significantly reduced proximity events between the two proteins in GRK2 - / - cells . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bar , 10 µm .

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(D) Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in GRK2-/- fibroblasts. Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in GRK2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bar, 10 µm.


( A ) Control ( + / + ) and Grk2 - / - NIH3T3 cells were transfected with the TOPflash Firefly ( F ) luciferase vector together with a control Renilla ( R ) luciferase vector , treated with Wnt3A ( 100 ng / ml ) for 24 hours and subjected to dual - luciferase assay . Note the impaired response to Wnt3A in Grk2 - / - cells . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated .

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(A) Control (+/+) and Grk2-/- NIH3T3 cells were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, treated with Wnt3A (100 ng/ml) for 24 hours and subjected to dual-luciferase assay. Note the impaired response to Wnt3A in Grk2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


( B ) Control ( + / + ) and Grk2 - / - NIH3T3 cells were treated with 40 ng / ml Wnt3A for 1 hour . Note the lower LRP6 expression and inhibited Wnt3A - induced LRP6 phosphorylation in the Grk2 - / - NIH3T3 cells . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated .

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(B) Control (+/+) and Grk2-/- NIH3T3 cells were treated with 40 ng/ml Wnt3A for 1 hour. Note the lower LRP6 expression and inhibited Wnt3A-induced LRP6 phosphorylation in the Grk2-/- NIH3T3 cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.


Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101 . Control human R00 - 082 chondrocytes ( C ) were treated with 20 µM CMPD101 overnight and then treated with 100 ng / ml Wnt3A for 1 hour . Note the less LRP6 phosphorylation ( pS1490 - and pT1572 - LRP6 ) in cells treated with CMPD101 . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated .

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Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. Control human R00-082 chondrocytes (C) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101 . rat chondrosarcoma ( RCS ) cells ( D ) were treated with 20 µM CMPD101 overnight and then treated with 100 ng / ml Wnt3A for 1 hour . Note the less LRP6 phosphorylation ( pS1490 - and pT1572 - LRP6 ) in cells treated with CMPD101 . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated .

Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. rat chondrosarcoma (RCS) cells (D) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in Grk2 - / - NIH3T3 cells . ( E ) Proximity ligation assay ( PLA ) between expressed FZD4 - GFP and ARRB2 - Flag showed significantly reduced proximity events between the two proteins in Grk2 - / - cells . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bars , 10 µm .

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Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in Grk2-/- NIH3T3 cells. (E) Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in Grk2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bars, 10 µm.


Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in Grk2 - / - NIH3T3 cells . ( F ) GRK2 addback rescued the interaction . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bars , 10 µm .

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Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in Grk2-/- NIH3T3 cells. (F) GRK2 addback rescued the interaction. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bars, 10 µm.


Box - plot showing the enrichment scores ( E - scores ) of all possible 8 - mers containing the G - box ( CACGTG ) , PBE - box ( CATGTG ) , and BRRE elements ( CGTGC / TG ) . Boxes represent the 25 - 75 % quartiles and the black line the median of distribution . Bars indicate the 1 - 25 % ( above ) and 75 - 100 % ( below ) quartiles . E - scores above 0 . 4 denote that binding of the proteins towards the indicated DNA element is statistically significant . Dashed blue line indicates the 0 . 4

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Box-plot showing the enrichment scores (E-scores) of all possible 8-mers containing the G-box (CACGTG), PBE-box (CATGTG), and BRRE elements (CGTGC/TG). Boxes represent the 25-75% quartiles and the black line the median of distribution. Bars indicate the 1-25% (above) and 75-100% (below) quartiles. E-scores above 0.4 denote that binding of the proteins towards the indicated DNA element is statistically significant. Dashed blue line indicates the 0.4 threshold


Protein - binding microarrays ( PBM ) were probed with PIF4 - His , MBP - BES1 and the PIF4 - His / MBP - BES1 complex . For PIF4 - BES1 complex purification , both proteins were co - expressed in E . coli using the double expression pCOLADuet - 1 vector . An anti - MBP antibody was used for BES1 and BES1 - PIF4 signal detection , while PIF4 was detected with an anti - His antibody Electrophoretic mobility shift assays ( EMSA ) showing interaction of the PIF4 , BES1 , and PIF4 - BES1 proteins with the conserved G - box , PBE - , and BRRE - elements in the PIL1 ( At2g46970 ) , PRE5 ( At3g28857 ) , and DWF4 ( At3g50660 ) promoters . Increasing amounts of protein were used for the

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Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody Electrophoretic mobility shift assays (EMSA) showing interaction of the PIF4, BES1, and PIF4-BES1 proteins with the conserved G-box, PBE-, and BRRE-elements in the PIL1 (At2g46970), PRE5 (At3g28857), and DWF4 (At3g50660) promoters. Increasing amounts of protein were used for the assay


Protein - binding microarrays ( PBM ) were probed with PIF4 - His , MBP - BES1 and the PIF4 - His / MBP - BES1 complex . For PIF4 - BES1 complex purification , both proteins were co - expressed in E . coli using the double expression pCOLADuet - 1 vector . An anti - MBP antibody was used for BES1 and BES1 - PIF4 signal detection , while PIF4 was detected with an anti - His antibody BES1 binds both BRRE - and G - box elements as a homodimer . A deletion of BES1 ( delN ) fused to MBP ( MBP - delN ) and the complete protein ( MBP - BES1 ) were co - expressed in E . coli . Formation of intermediate mobility bands , indicative of DNA recognition by a dimeric form of the protein was tested in EMSA assays . A signal corresponding to the dimeric full - length MBP - BES1 and MBP - delN complex was detected with both DNA probes . Increasing amounts of the proteins were used in the

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Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody BES1 binds both BRRE- and G-box elements as a homodimer. A deletion of BES1 (delN) fused to MBP (MBP-delN) and the complete protein (MBP-BES1) were co-expressed in E.coli. Formation of intermediate mobility bands, indicative of DNA recognition by a dimeric form of the protein was tested in EMSA assays. A signal corresponding to the dimeric full-length MBP-BES1 and MBP-delN complex was detected with both DNA probes. Increasing amounts of the proteins were used in the assay


Co - expression of the PIF4 and BES1 effector constructs leads to synergistic pPIL1 activation , and reverses BES1 - dependent inhibition of the pDWF4 reporter . The pPIL1 and pDWF4 promoters including three G - boxes ( green boxes ) and two BRRE - elements ( orange boxes ) were fused to the firefly luciferase reporter gene ( LUC ) and co - transfected with 35S : : PIF4 , 35S : : BES1 and 35S : : bes1 - D effector constructs into Nicotiana benthamiana leaves . Leaf discs were collected 48 hours after infiltration and luciferase activity was measured in a microplate luminometer .

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Co-expression of the PIF4 and BES1 effector constructs leads to synergistic pPIL1 activation, and reverses BES1-dependent inhibition of the pDWF4 reporter. The pPIL1 and pDWF4 promoters including three G-boxes (green boxes) and two BRRE- elements (orange boxes) were fused to the firefly luciferase reporter gene (LUC) and co-transfected with 35S::PIF4, 35S::BES1 and 35S::bes1-D effector constructs into Nicotiana benthamiana leaves. Leaf discs were collected 48 hours after infiltration and luciferase activity was measured in a microplate luminometer.


Heat map and fold - change graph representation of the genes identified in A . Expression data were obtained from Genevestigator and heatmap and clustering analyses were performed using the MeV software . PIF + BES - UP indicates genes up - regulated by PIF and BES and identified as targets of both the PIF and BES factors . BES - DOWN are BES repressed targets , while BES - UP correspond to BES induced targets . The PIF - UP cluster includes PIF induced genes directly bound by the PIF factors .

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Heat map and fold-change graph representation of the genes identified in A. Expression data were obtained from Genevestigator and heatmap and clustering analyses were performed using the MeV software. PIF+BES-UP indicates genes up-regulated by PIF and BES and identified as targets of both the PIF and BES factors. BES-DOWN are BES repressed targets, while BES-UP correspond to BES induced targets. The PIF-UP cluster includes PIF induced genes directly bound by the PIF factors.


Time course analysis of BES1 protein accumulation levels . pBES1 : : BES1 - GFP seedlings were grown under short days for 6 days and samples were harvested each 2 hours for a 24 h interval . Total protein extracts were used for western blot analysis with an anti - GFP antibody to detect the BES1 - GFP protein . Hybridization with anti - RPT5 antibody is included as a loading control . Dark and light periods are represented as black and white bars , respectively . The experiment was repeated twice with similar

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Time course analysis of BES1 protein accumulation levels. pBES1::BES1-GFP seedlings were grown under short days for 6 days and samples were harvested each 2 hours for a 24 h interval. Total protein extracts were used for western blot analysis with an anti-GFP antibody to detect the BES1-GFP protein. Hybridization with anti-RPT5 antibody is included as a loading control. Dark and light periods are represented as black and white bars, respectively. The experiment was repeated twice with similar results


Diurnal oscillation of BES1 and BZR1 transcripts . Col - 0 seedlings were grown for 6 days under short days and samples were harvested every 3 hours . BES1 and BZR1 expression levels were determined by qRT - PCR analyses and normalized to PP2A . Relative levels are the mean of three technical replicates and error bars represent standard deviation . The experiment was performed three times with similar results . Dark periods are indicated by

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Diurnal oscillation of BES1 and BZR1 transcripts. Col-0 seedlings were grown for 6 days under short days and samples were harvested every 3 hours. BES1 and BZR1 expression levels were determined by qRT-PCR analyses and normalized to PP2A. Relative levels are the mean of three technical replicates and error bars represent standard deviation. The experiment was performed three times with similar results. Dark periods are indicated by shading


Western blot analyses of 35S : : BES - GFP seedlings grown for six days under short days . Samples were collected every two hours , starting two hours before lights on ( ZT - 2 ) to ZT6 . Western blots were hybridized with an anti - GFP antibody to detect the BES1 - GFP protein , and anti - RPT5 as a loading

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Western blot analyses of 35S::BES-GFP seedlings grown for six days under short days. Samples were collected every two hours, starting two hours before lights on (ZT-2) to ZT6. Western blots were hybridized with an anti-GFP antibody to detect the BES1-GFP protein, and anti-RPT5 as a loading control


BES1 but not BZR1 is stabilized in the light . 35S : : BES1 - GFP and 35S : : BZR1 - GFP seedlings were grown for 6 days in continuous white light ( WL ) , red light ( RL ) , and continuous dark ( DARK ) , and the BES1 and BZR1 proteins analyzed by western blot using an anti - GFP antibody . Hybridization with an anti - RPT5 antibody was used as a loading

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BES1 but not BZR1 is stabilized in the light. 35S::BES1-GFP and 35S::BZR1-GFP seedlings were grown for 6 days in continuous white light (WL), red light (RL), and continuous dark (DARK), and the BES1 and BZR1 proteins analyzed by western blot using an anti-GFP antibody. Hybridization with an anti-RPT5 antibody was used as a loading control


BES1 accumulation is independent of brassinosteroid levels . Six day - old 35S : : BES - GFP seedlings were treated overnight with 100 nM epi - brassinolide ( BL ) or 0 . 5 µM brassinazole ( BRZ ) . Material was collected at the indicated time points and analyzed by western blot hybridization with anti - GFP and - RPT5

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BES1 accumulation is independent of brassinosteroid levels. Six day-old 35S::BES-GFP seedlings were treated overnight with 100 nM epi-brassinolide (BL) or 0.5 µM brassinazole (BRZ). Material was collected at the indicated time points and analyzed by western blot hybridization with anti-GFP and -RPT5 antibodies


In vivo analysis of PIF4 - BES1 complex formation . Six day - old pPIF4 : : PIF4 - HA seedlings grown under short day conditions were collected at ZT0 , ZT1 and ZT8 . PIF4 - HA was immunoprecipitated with an anti - HA antibody and the pulled - down BES1 protein immunodetected with an anti - BES antibody . * Indicates a non - specific band by the primary antibody used to immunoprecipitation .

In vivo analysis of PIF4-BES1 complex formation. Six day-old pPIF4::PIF4-HA seedlings grown under short day conditions were collected at ZT0, ZT1 and ZT8. PIF4-HA was immunoprecipitated with an anti-HA antibody and the pulled-down BES1 protein immunodetected with an anti-BES antibody. * Indicates a non-specific band by the primary antibody used to immunoprecipitation.


Col - 0 ( black ) , PIF4 - OX ( red ) and pifq ( blue ) seedlings were grown for 6 days under short days and samples harvested at the indicated time points . Total RNA was used for qRT - PCR quantification of PIF + BES - UP and the BR biosynthetic BES - DOWN transcripts , using PP2A as endogenous control . Error bars represent SD of three technical replicates . Dark periods are shown in grey . Relative expression of the XTR7 , PRE5 , IAA19 and BR6ox2 genes in PIF4 - OX lines are referred to the right y axis of the graph . The experiment was repeated three times with similar results .

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Col-0 (black), PIF4-OX (red) and pifq (blue) seedlings were grown for 6 days under short days and samples harvested at the indicated time points. Total RNA was used for qRT-PCR quantification of PIF+BES-UP and the BR biosynthetic BES-DOWN transcripts, using PP2A as endogenous control. Error bars represent SD of three technical replicates. Dark periods are shown in grey. Relative expression of the XTR7, PRE5, IAA19 and BR6ox2 genes in PIF4-OX lines are referred to the right y axis of the graph. The experiment was repeated three times with similar results.


Col - 0 and pifq seedlings were grown under short days at 22ºC and 28ºC , and samples were collected at the indicated time points . Expression of BR biosynthetic genes was analyzed by qRT - PCR , using the constitutive PP2A gene as internal control . Error bars represent SD of three technical replicates . The experiment was repeated twice with similar

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

Col-0 and pifq seedlings were grown under short days at 22ºC and 28ºC, and samples were collected at the indicated time points. Expression of BR biosynthetic genes was analyzed by qRT-PCR, using the constitutive PP2A gene as internal control. Error bars represent SD of three technical replicates. The experiment was repeated twice with similar results


Activation of the XTR7 PIF + BES - UP target was studied as a positive control of the experiment . Error bars represent SD of three technical replicates . Dark periods are shown in grey . The experiment was performed three times with similar

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

Activation of the XTR7 PIF+BES-UP target was studied as a positive control of the experiment. Error bars represent SD of three technical replicates. Dark periods are shown in grey. The experiment was performed three times with similar results


Increased levels of de - phosphorylated BES1 at elevated temperatures depend on PIFs activation of BR synthesis . Col - 0 and pifq seedlings were grown for 6 days under short days and 22ºC or 28ºC , on half strength MS media ( mock ) or media supplemented with 0 . 5 µM BRZ . BES1 was analyzed by western blot hybridization with an anti - BES1 antibody . RPT5 was used as a loading control .

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Increased levels of de-phosphorylated BES1 at elevated temperatures depend on PIFs activation of BR synthesis. Col-0 and pifq seedlings were grown for 6 days under short days and 22ºC or 28ºC, on half strength MS media (mock) or media supplemented with 0.5 µM BRZ. BES1 was analyzed by western blot hybridization with an anti-BES1 antibody. RPT5 was used as a loading control.


( a - d ) Degradation of long - lived proteins in MEFs from wild - type ( 18Q - htt ) and mutant huntingtin knock - in ( 111Q - htt ) mice . ( a , b ) Rates of protein degradation after serum removal ( a )

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(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after serum removal (a)


( a - d ) Degradation of long - lived proteins in MEFs from wild - type ( 18Q - htt ) and mutant huntingtin knock - in ( 111Q - htt ) mice . ( a , b ) Rates of protein degradation after treatment with rapamycin ( rapam ) or thapsigargin ( thapsig ) ( b ) .

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(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after treatment with rapamycin (rapam) or thapsigargin (thapsig) (b).


( c ) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl .

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(c) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl.


( d ) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3 - methyladenine ( 3 - MA ) .

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(d) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3-methyladenine (3-MA).


( e ) Degradation of long - lived proteins in striatal cells from wild - type ( 7Q - htt ) and mutant huntingtin knock - in mice ( 111Q - htt ) in response to different autophagic stimuli .

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(e) Degradation of long-lived proteins in striatal cells from wild-type (7Q-htt) and mutant huntingtin knock-in mice (111Q-htt) in response to different autophagic stimuli.


( f , g ) Rates of protein degradation in wild - type and HD MEFs ( f ) and striatal cells ( g ) in the absence ( control ) or presence ( Atg7 − ) of Atg7 RNA interference .

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(f,g) Rates of protein degradation in wild-type and HD MEFs (f) and striatal cells (g) in the absence (control) or presence (Atg7−) of Atg7 RNA interference.


( h ) Contribution of macroautophagy ( macroaut ) to the degradation of long - lived proteins in lymphoblasts from unaffected controls ( UC ) and people with HD . Mean + s . d . of values from two to four different individuals and three or four different experiments . * P 0 . 05 versus untreated , # P 0 . 05 versus control .

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(h) Contribution of macroautophagy (macroaut) to the degradation of long-lived proteins in lymphoblasts from unaffected controls (UC) and people with HD. Mean + s.d. of values from two to four different individuals and three or four different experiments. *P 0.05 versus untreated, #P 0.05 versus control.


( a ) Top : LC3 immunostaining of 18Q - htt and 111Q - htt MEFs maintained in the presence ( + ) or absence ( - ) of serum and inhibitors of lysosomal proteolysis ( inhib + ) . Bottom : mean number of LC3 + vesicles per cell in cells maintained in the presence ( left ) or absence ( right ) of serum . n = 4 .

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(a) Top: LC3 immunostaining of 18Q-htt and 111Q-htt MEFs maintained in the presence (+) or absence (-) of serum and inhibitors of lysosomal proteolysis (inhib+). Bottom: mean number of LC3+ vesicles per cell in cells maintained in the presence (left) or absence (right) of serum. n = 4.


( b ) Top : LC3 immunoblot of the same cells after serum ( S ) removal or thapsigargin ( TG ) treatment . PI , protease inhibitors . Bottom : LC3 - II levels and LC3 - II flux ( calculated as a multiple of LC3 - II value in absence of PI ; lateral numbers ) . n = 4 .

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(b) Top: LC3 immunoblot of the same cells after serum (S) removal or thapsigargin (TG) treatment. PI, protease inhibitors. Bottom: LC3-II levels and LC3-II flux (calculated as a multiple of LC3-II value in absence of PI; lateral numbers). n = 4.


( c ) LC3 - II values and LC3 - II flux in neuronal cultures from wild - type ( WT ) or HD94 mice ( HD ) grown on wild - type rat astrocyte monolayers , analyzed as in b . n = 4 .

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(c) LC3-II values and LC3-II flux in neuronal cultures from wild-type (WT) or HD94 mice (HD) grown on wild-type rat astrocyte monolayers, analyzed as in b. n = 4.


( d ) LC3 staining of lymphoblasts , from three unaffected controls ( UC ) and people with HD , maintained in the presence or absence of serum . Accession numbers are indicated above panels . Right : mean number of LC3 + vesicles . Extended study in Supplementary Figure 8b , c .

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(d) LC3 staining of lymphoblasts, from three unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Accession numbers are indicated above panels. Right: mean number of LC3+ vesicles. Extended study in Supplementary Figure 8b,c.


( e ) Immunoblots for LC3 - II in UC and HD lymphoblasts treated or not treated with protease inhibitors . Right : LC3 - II values and LC3 - II flux . Extended study in Supplementary Figure 8a . Mean + s . d . ; # P 0 . 05 . Full - length blots in Supplementary Figure 20 .

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(e) Immunoblots for LC3-II in UC and HD lymphoblasts treated or not treated with protease inhibitors. Right: LC3-II values and LC3-II flux. Extended study in Supplementary Figure 8a. Mean + s.d.; #P 0.05. Full-length blots in Supplementary Figure 20.


( a ) Electron microscopy of striatal neurons from control and HD94 mice grown over a wild - type ratastrocyte monolayer . Higher magnification fields show the double membrane and clear content of the cytosolic vesicles . Top right : number of vacuoles per cell profile ( 13 - 17 cell profiles in triplicate experiments ; * P 0 . 05 ) . Arrows , enlarged electron - clear vesicles .

(a) Electron microscopy of striatal neurons from control and HD94 mice grown over a wild-type ratastrocyte monolayer. Higher magnification fields show the double membrane and clear content of the cytosolic vesicles. Top right: number of vacuoles per cell profile (13-17 cell profiles in triplicate experiments; *P 0.05). Arrows, enlarged electron-clear vesicles.


( b ) Electron micrographs of striatal cells from mutant huntingtin knock - in ( 111Q - htt ) and wild - type ( 7Q - htt ) mice .

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(b) Electron micrographs of striatal cells from mutant huntingtin knock-in (111Q-htt) and wild-type (7Q-htt) mice.


( c ) Electron micrographs of lymphoblasts from unaffected controls ( UC ) and people with HD , maintained in the presence or absence of serum . Arrows , enlarged electron - clear vesicles .

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(c) Electron micrographs of lymphoblasts from unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Arrows, enlarged electron-clear vesicles.


( d ) Immunogold for LC3 in striatal cells from mutant huntingtin knock - in mice ( 111Q - htt ) , striatal neurons from HD94 mice and lymphoblasts ( lymph ) from people with HD . Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11 .

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(d) Immunogold for LC3 in striatal cells from mutant huntingtin knock-in mice (111Q-htt), striatal neurons from HD94 mice and lymphoblasts (lymph) from people with HD. Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11.


( a ) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q - htt and 111Q - htt mice . Insets : higher magnification of single vesicles . Right : percentage of vesicles with electron - clear ( light ) , vesiculated ( multivesicular ) or electron - dense ( dark ) contents . Mean + s . e . m . of three different isolations ( > ∼ 1 , 000 autophagic vacuoles ) .

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(a) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q-htt and 111Q-htt mice. Insets: higher magnification of single vesicles. Right: percentage of vesicles with electron-clear (light), vesiculated (multivesicular) or electron-dense (dark) contents. Mean + s.e.m. of three different isolations (> ∼1,000 autophagic vacuoles).


( b ) Bidimensional electrophoresis and Sypro Ruby staining of these fractions . Left , intact vacuoles ; right , vacuole contents . Horizontal dimension , isoelectric point ( pH ) ; vertical , molecular mass ( kDa ) . * P 0 . 05 .

(b) Bidimensional electrophoresis and Sypro Ruby staining of these fractions. Left, intact vacuoles; right, vacuole contents. Horizontal dimension, isoelectric point (pH); vertical, molecular mass (kDa). *P 0.05.


( a ) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes ( APHL ) isolated from livers of 18Q - htt ( 18Q ) and 111Q - htt ( 111Q ) mice . Cyt , cytochrome ; Dyn IC , dynein intermediate chain ; ADRP , adipose differentiation - related protein ; Polyubq , polyubiquitin . Bottom : amount of each protein in 111Q - htt sample , as a multiple of its amount in 18Q - htt sample . Mean + s . d . ; n = 4 .

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(a) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes (APHL) isolated from livers of 18Q-htt (18Q) and 111Q-htt (111Q) mice. Cyt, cytochrome; Dyn IC, dynein intermediate chain; ADRP, adipose differentiation-related protein; Polyubq, polyubiquitin. Bottom: amount of each protein in 111Q-htt sample, as a multiple of its amount in 18Q-htt sample. Mean + s.d.; n = 4.


( b ) Immunoblots for htt and LC3 in homogenates ( Homog ) , cytosol and fractions enriched in APHs and enriched in autophagolysosomes ( APHL ) isolated from livers of wild - type ( 18Q - htt ) and mutant huntingtin knock - in mice ( 111Q - htt ) . Representative one of four experiments with duplicated samples . * P 0 . 05 . Full - length blots in Supplementary Figure 20 .

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(b) Immunoblots for htt and LC3 in homogenates (Homog), cytosol and fractions enriched in APHs and enriched in autophagolysosomes (APHL) isolated from livers of wild-type (18Q-htt) and mutant huntingtin knock-in mice (111Q-htt). Representative one of four experiments with duplicated samples. *P 0.05. Full-length blots in Supplementary Figure 20.


( a ) Immunoblot for htt or p62 from total APHs ( T ) and their corresponding membranes ( Mbr ) and matrices ( Mtx ) isolated from livers of wild - type htt ( 18Q ) and 111Q - htt mutant huntingtin knock - in mice ( 111Q ) . Left , representative immunoblots . Right , distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots . AV , autophagic vacuoles . Mean + s . d . * P 0 . 05 compared to wild - type values .

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(a) Immunoblot for htt or p62 from total APHs (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from livers of wild-type htt (18Q) and 111Q-htt mutant huntingtin knock-in mice (111Q). Left, representative immunoblots. Right, distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots. AV, autophagic vacuoles. Mean + s.d. *P 0.05 compared to wild-type values.


( b ) Filter retardation analysis of the same fractions as in a , blotted for htt . Negative signal indicated absence of aggregates retained in the filter .

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(b) Filter retardation analysis of the same fractions as in a, blotted for htt. Negative signal indicated absence of aggregates retained in the filter.


( c ) Immunoblot for ubiquitin in homogenates ( Homog ) , cytosol and the autophagic fractions described in a , using 6 % ( top ) and 16 % ( bottom ) gels . Dotted line separates stacking and running parts of gel .

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(c) Immunoblot for ubiquitin in homogenates (Homog), cytosol and the autophagic fractions described in a, using 6% (top) and 16% (bottom) gels. Dotted line separates stacking and running parts of gel.


( d ) Immunoblot for htt , LC3 and p62 , of brain homogenates ( Homog ) and fractions enriched in APHs from the same mouse groups as in a .

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(d) Immunoblot for htt, LC3 and p62, of brain homogenates (Homog) and fractions enriched in APHs from the same mouse groups as in a.


( e ) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co - immunoprecipitation for htt under low stringency . Input for 111Q - htt autophagic vacuoles was one - third of that for 18Q - htt to avoid antibody saturation . Levels of htt ( top ) and p62 ( bottom ) in the input , immunoprecipitate ( IP ) and flow through ( FT ) are shown . Full - length blots in Supplementary Figure 21 .

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(e) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co-immunoprecipitation for htt under low stringency. Input for 111Q-htt autophagic vacuoles was one-third of that for 18Q-htt to avoid antibody saturation. Levels of htt (top) and p62 (bottom) in the input, immunoprecipitate (IP) and flow through (FT) are shown. Full-length blots in Supplementary Figure 21.


( a ) Neutral lipids in MEFs from 18Q - htt and 111Q - htt mice stained with BODIPY 493 / 503 .

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(a) Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503.


( b ) Electron micrographs of livers from 18Q - htt and 111Q - htt mice . Right : number of lipid droplets ( LD ) per cell profile ( prof . ) , mean area of LDs and percentage of cellular area occupied by LDs . Mean + s . d . ; n = 3 .

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(b) Electron micrographs of livers from 18Q-htt and 111Q-htt mice. Right: number of lipid droplets (LD) per cell profile (prof.), mean area of LDs and percentage of cellular area occupied by LDs. Mean + s.d.; n = 3.


( c - e ) Neutral lipids in striatal cells from 7Q - htt and 111Q - httmice ( c ) , grown on a monolayer of their own

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(c-e) Neutral lipids in striatal cells from 7Q-htt and 111Q-httmice (c), grown on a monolayer of their own astrocytes


( c - e ) Neutral lipids in primary striatal neurons from 18Q - htt and 111Q - httmice grown on a monolayer of their own astrocytes ( d )

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(c-e) Neutral lipids in primary striatal neurons from 18Q-htt and 111Q-httmice grown on a monolayer of their own astrocytes (d)


( c - e ) Neutral lipids in lymphoblasts from a control ( UC ) and HD - affected human ( e ) stained with BODIPY 493 / 503 . MAP2 staining highlights neurons . Extended study in Supplementary Figure 15b .

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(c-e) Neutral lipids in lymphoblasts from a control (UC) and HD-affected human (e) stained with BODIPY 493/503. MAP2 staining highlights neurons. Extended study in Supplementary Figure 15b.


( f ) Electron micrographs of a lymphoblast from an individual with HD ( with 78 and 15 polyglutamine repeats in the two alleles ) . Green arrows , LDs .

(f) Electron micrographs of a lymphoblast from an individual with HD (with 78 and 15 polyglutamine repeats in the two alleles). Green arrows, LDs.


( g ) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects . Numbers of polyglutamine repeats are shown below . Mean + s . d . of > 100 cell profiles . Dotted red line , mean value of the respective samples . * P 0 . 05 .

(g) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects. Numbers of polyglutamine repeats are shown below. Mean + s.d. of >100 cell profiles. Dotted red line, mean value of the respective samples. *P 0.05.


( h ) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals ( top ) and two individuals with HD ( bottom ) . Nuclei are highlighted with hematoxylin . Lipid droplets are visible as red puncta . Subject identifiers are shown in each panel .

(h) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals (top) and two individuals with HD (bottom). Nuclei are highlighted with hematoxylin. Lipid droplets are visible as red puncta. Subject identifiers are shown in each panel.


( i ) The percentage of total cellular area occupied by lipid droplets ( left ) and the average number of lipid droplets per cell ( right ) was calculated for each of the samples by quantification of eight or nine different fields . * P 0 . 001 ; analysis of variance , P 0 . 01 for each person with HD versus each control individual by Tukey post hoc test . Subject identifiers are as in h .

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(i) The percentage of total cellular area occupied by lipid droplets (left) and the average number of lipid droplets per cell (right) was calculated for each of the samples by quantification of eight or nine different fields. *P 0.001; analysis of variance, P 0.01 for each person with HD versus each control individual by Tukey post hoc test. Subject identifiers are as in h.


( a ) Top : immunofluorescence for the mitochondrial marker COX IV in 18Q - htt and 111Q - htt MEFs maintained in the presence or absence of serum . Bottom : number of mitochondria per cell . Mean + s . d . of 10 - 20 cells in three different experiments .

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(a) Top: immunofluorescence for the mitochondrial marker COX IV in 18Q-htt and 111Q-htt MEFs maintained in the presence or absence of serum. Bottom: number of mitochondria per cell. Mean + s.d. of 10-20 cells in three different experiments.


( b ) Electron micrographs of striatal neurons from wild - type ( Ctr ) and HD94 mice grown over a wild - type ratastrocyte monolayer . Arrows , abnormally short ( black ) or abnormally long ( green ) mitochondria . Right : number of mitochondria per cell profile . Mean + s . d . of ten cells per group in triplicates .

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(b) Electron micrographs of striatal neurons from wild-type (Ctr) and HD94 mice grown over a wild-type ratastrocyte monolayer. Arrows, abnormally short (black) or abnormally long (green) mitochondria. Right: number of mitochondria per cell profile. Mean + s.d. of ten cells per group in triplicates.


( c ) Left : electron micrographs of lymphoblasts from controls ( UC ) or people with HD . Arrows as in b . Right : percentage of cellular area occupied by mitochondria in different individuals . Line indicates population mean .

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(c) Left: electron micrographs of lymphoblasts from controls (UC) or people with HD. Arrows as in b. Right: percentage of cellular area occupied by mitochondria in different individuals. Line indicates population mean.


( d , e ) Striatal cells from 7Q - htt and 111Q - htt mice ( d ) stained with Mitotracker and Mito - ROS . Right , merged images . Percentage of colocalization is indicated at the bottom in

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(d,e) Striatal cells from 7Q-htt and 111Q-htt mice (d) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d


MEFs from 18Q - htt and 111Q - httmice ( e ) stained with Mitotracker and Mito - ROS . Right , merged images . Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e . CCCP was added to control cells in e as a positive control for depolarization . * P 0 . 05 .

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MEFs from 18Q-htt and 111Q-httmice (e) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e. CCCP was added to control cells in e as a positive control for depolarization. *P 0.05.


( f , g ) Striatal cells from 7Q - htt and 111Q - htt mice untreated or treated with vinblastine were co - stained for LC3 and Mitotracker ( f ) . Arrows , colocalization events . Extended study in Supplementary Figure 16 .

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(f,g) Striatal cells from 7Q-htt and 111Q-htt mice untreated or treated with vinblastine were co-stained for LC3 and Mitotracker (f). Arrows, colocalization events. Extended study in Supplementary Figure 16.


( f , g ) Striatal cells from 7Q - htt and 111Q - httmice untreated or treated with vinblastine were co - stained for LC3 and BODIPY 493 / 503 ( g ) . Arrows , colocalization events . Extended study in Supplementary Figure 16 .

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(f,g) Striatal cells from 7Q-htt and 111Q-httmice untreated or treated with vinblastine were co-stained for LC3 and BODIPY 493/503 (g). Arrows, colocalization events. Extended study in Supplementary Figure 16.


HeLa cells overexpressing the HA - tagged MICU1 WT , MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 ( mitochondrial marker ) . Merged images are indicated ( merge ) . Scale bar 10

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HeLa cells overexpressing the HA-tagged MICU1 WT, MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm


Representative images of the 2mt - GCaMP6m 474 / 410 ratio of ShMICU1 HeLa stable cells expressing an empty vector ( ctrl ) or the MICU1 WT , MICU1 SD , and MICU1 SA . Scale bar 10

Representative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μm


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in ShRNA control ( plko ) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs ( n = 5 independent experiments ; 55 - 67cells )

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=5 independent experiments; 55-67cells)


Representative kinetics ( E ) and analysis ( F ) of aequorin - based [ Ca2 + ] m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2 , 5 - di - tert - butylhydroquinone ( TBHQ ) in the absence of extracellular Ca2 + ( n = 3 independent experiments )

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Representative kinetics (E) and analysis (F) of aequorin-based [Ca2+]m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2,5-di-tert-butylhydroquinone (TBHQ) in the absence of extracellular Ca2+ (n=3 independent experiments)


Representative kinetics ( G ) and analysis ( H ) of aequorin - based [ Ca2 + ] m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid ( CPA ) in the presence of 100 μM EGTA ( n = 3 independent experiments )

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Representative kinetics (G) and analysis (H) of aequorin-based [Ca2+]m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)


Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR / Cas9 - mediated genome editing . The results for 12 of the 36 clones that were examined are

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

Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR/Cas9-mediated genome editing. The results for 12 of the 36 clones that were examined are shown


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial - targeted GCaMP6m , in MICU1 - KO cells generated using the CRISPR / Cas9 technique and transfected with the indicated constructs ( n = 3 independent experiments ; 30 - 56 cells )

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in MICU1-KO cells generated using the CRISPR/Cas9 technique and transfected with the indicated constructs (n=3 independent experiments; 30-56 cells)


Representative kinetics ( K ) and analysis ( L ) of aequorin - based [ Ca2 + ] m measurements in intact MICU1 - KO cells generated using the CRISPR / Cas9 technique , transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2 + ( n = 3 independent experiments . )

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Representative kinetics (K) and analysis (L) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2+ (n=3 independent experiments.)


Representative kinetics ( M ) and analysis ( N ) of aequorin - based [ Ca2 + ] m measurements in intact MICU1 - KO cells generated using the CRISPR / Cas9 technique , transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA ( n = 3 independent experiments ) .

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Representative kinetics (M) and analysis (N) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA (n=3 independent experiments).


HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated ( S473 ) Akt ( p - Akt ) or HSP60 ( mitochondrial marker ) . Merged images are indicated ( merge ) . Scale bar 10

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HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm


Analysis of the number of cells , expressed as a percentage , showing obvious mitochondrial staining of activated ( S473 phosphorylated ) Akt ( p - Akt ) ( n = 3 independent experiments ; 350 - 355 cells )

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Analysis of the number of cells, expressed as a percentage, showing obvious mitochondrial staining of activated (S473 phosphorylated) Akt (p-Akt) (n=3 independent experiments; 350-355 cells)


Hek293T cells treated with vehicle or 1 μM rapamycin ( Rapa . ) for 4 h were fractionated into Cytosol ( Cyt . ) or mitochondrial ( Mito . ) extracts and analyzed by western blotting . E - cadherin : plasma membrane marker ; β - tubulin : cytosolic marker ; VDAC : mitochondrial marker ; Homo : cell

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Hek293T cells treated with vehicle or 1 μM rapamycin (Rapa.) for 4 h were fractionated into Cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate


Mouse livers treated with vehicle or 1 μM rapamycin ( Rapa . ) for 16 h by an intraperitoneal injection were fractionated into cytosol ( Cyt . ) or mitochondrial ( Mito . ) extracts and analyzed by western blotting . Phosphorylated ( S2468 ) mTOR ( p - mTOR ) was used to assess the rapamycin activity . E - cadherin : plasma membrane marker ; β - tubulin : cytosolic marker ; VDAC : mitochondrial marker ; Homo : cell

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Mouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate


Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt , phosphorylated ( S473 ) Akt ( p - Akt ) , and mitochondrial proteins with known localizations . Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture . OMM : outer mitochondrial membrane ; IMS : intermembrane

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

Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt, phosphorylated (S473) Akt (p-Akt), and mitochondrial proteins with known localizations. Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture. OMM: outer mitochondrial membrane; IMS: intermembrane space


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of vehicle - or rapamycin ( Rapa . , 1 μM for 4 h ) - treated Hek293T cell mitochondria following carbonate extraction at pH 11 . 5 . Akt , phosphorylated ( S473 ) Akt ( p - Akt ) , MICU1 , the established integral membrane proteins TIM23 and MCU , and the soluble protein cytochrome c ( Cyt . c ) was

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of vehicle- or rapamycin (Rapa., 1 μM for 4 h)-treated Hek293T cell mitochondria following carbonate extraction at pH 11.5. Akt, phosphorylated (S473) Akt (p-Akt), MICU1, the established integral membrane proteins TIM23 and MCU, and the soluble protein cytochrome c (Cyt. c) was analyzed


Hek293T cells were transfected with the GFP - tagged wild - type ( WT ) MICU1 and then treated with 1 μM rapamycin ( Rapa . ) alone for 4 h or in combination with the Akt inhibitor triciribine ( 10 μM ) . GFP - MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) and phosphorylated ( S473 ) Akt ( p - Akt ) antibodies by western

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Hek293T cells were transfected with the GFP-tagged wild-type (WT) MICU1 and then treated with 1 μM rapamycin (Rapa.) alone for 4 h or in combination with the Akt inhibitor triciribine (10 μM). GFP-MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) and phosphorylated (S473) Akt (p-Akt) antibodies by western blotting


Hek293T cells were transfected with either the GFP - tagged wild - type ( WT ) MICU1 or MICU1 S124A - GFP mutant . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western blotting .

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Hek293T cells were transfected with either the GFP-tagged wild-type (WT) MICU1 or MICU1 S124A-GFP mutant. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting.


Mitochondrial preparations of both Hek293T ( left ) and HeLa ( right ) cells transiently transfected with an HA - tagged constitutively active Akt T308D / S473D ( Akt D / D ) construct were analyzed by western blotting . GAPDH : cytosolic marker ; TIM23 : mitochondrial

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Mitochondrial preparations of both Hek293T (left) and HeLa (right) cells transiently transfected with an HA-tagged constitutively active Akt T308D/S473D (Akt D/D) construct were analyzed by western blotting. GAPDH: cytosolic marker; TIM23: mitochondrial marker


Resting mitochondrial calcium levels in the control ( ctrl ) and Akt D / D - expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial - targeted GCaMP6m ( n = 4 independent experiments ; 44 - 49 cells )

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Resting mitochondrial calcium levels in the control (ctrl) and Akt D/D-expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 44-49 cells)


Schematic model of the HA - tagged constitutively active Akt form targeted to the mitochondrial intermembrane space ( mt - Akt D / D ) . Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence . TOM20 was used as a mitochondrial marker . Merged images are indicated ( merge ) . Scale bar 10

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Schematic model of the HA-tagged constitutively active Akt form targeted to the mitochondrial intermembrane space (mt-Akt D/D). Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence. TOM20 was used as a mitochondrial marker. Merged images are indicated (merge). Scale bar 10 μm


Hek293T cells were transfected with the indicated constructs . The GFP - tagged MICU1 was immunoprecipitated from the whole - cell lysate , and the precipitated proteins were immunoblotted with the HA and phosphorylated ( S473 ) Akt ( p - Akt )

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Hek293T cells were transfected with the indicated constructs. The GFP-tagged MICU1 was immunoprecipitated from the whole-cell lysate, and the precipitated proteins were immunoblotted with the HA and phosphorylated (S473) Akt (p-Akt) antibodies


Hek293T cells were transfected with the indicated constructs . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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Hek293T cells were transfected with the indicated constructs. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Resting mitochondrial calcium levels in control ( ctrl ) and mitochondrial - targeted Akt D / D ( mt - Akt D / D ) - expressing HeLa cells , evaluated through ratiometric imaging of the mitochondrial - targeted GCaMP6m ( n = 4 independent experiments ; 43 - 47 cells )

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Resting mitochondrial calcium levels in control (ctrl) and mitochondrial-targeted Akt D/D (mt-Akt D/D)-expressing HeLa cells, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 43-47 cells)


Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m ( n = 3 independent experiments ; 38 - 50 cells )

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Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m (n=3 independent experiments; 38-50 cells)


Representative kinetics ( H ) and analysis ( I ) of aequorin - based [ Ca2 + ] m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [ Ca2 + ] ( n = 3 independent experiments ) .

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Representative kinetics (H) and analysis (I) of aequorin-based [Ca2+]m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [Ca2+] (n=3 independent experiments).


Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs . pre - MICU1 : precursor form of MICU1 ; m - MICU1 : mature form of

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Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. pre-MICU1: precursor form of MICU1; m-MICU1: mature form of MICU1


Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs . Where indicated , the cells were treated with 5 μM MG132 for 4 h . pre : precursor ; int : intermediate ; m : mature

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Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. Where indicated, the cells were treated with 5 μM MG132 for 4 h. pre: precursor; int: intermediate; m: mature form


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs , following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markers . The cells were treated with 5 μM MG132 for 4 h before

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs, following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. The cells were treated with 5 μM MG132 for 4 h before fractionation


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT - HA or MICU1 SD - HA , together with MCU - flag and EMRE constructs , following carbonate extraction at pH 7 . 4 , pH 10 . 5 , pH 11 . 5 or pH 12 . 5 . The cells were treated with 5 μM MG132 for 4 h before

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT-HA or MICU1 SD-HA, together with MCU-flag and EMRE constructs, following carbonate extraction at pH 7.4, pH 10.5, pH 11.5 or pH 12.5. The cells were treated with 5 μM MG132 for 4 h before fractionation


ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs . Cells were incubated with 50 μg / ml cycloheximide ( CHX ) for the indicated times , collected and subjected to western blotting analysis as indicated . In the graph , the amount of HA ( MICU1 ) , both WT and mutants , is represented relative to the amount at time 0 . ( n = 3 independent experiments )

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ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1), both WT and mutants, is represented relative to the amount at time 0. (n=3 independent experiments)


ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs . Cells were harvested , and total protein was extracted and subjected to western blotting analysis with the indicated antibodies . Immunoblotting was performed under reducing ( with the presence of DTT ) or non - reducing ( non - red ) conditions ( without DTT ) . Cells were treated with 5 μM MG132 for 4 h before

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

ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were harvested, and total protein was extracted and subjected to western blotting analysis with the indicated antibodies. Immunoblotting was performed under reducing (with the presence of DTT) or non-reducing (non-red) conditions (without DTT). Cells were treated with 5 μM MG132 for 4 h before lysis


Quantification of HA levels in non - reducing condition showed in ( E ) . ( n = 3 independent experiments )

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Quantification of HA levels in non-reducing condition showed in (E). (n=3 independent experiments)


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs ( n = 3 independent experiments ; 46 - 54 cells ) .

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=3 independent experiments; 46-54 cells).


Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and / or IMMP2L . int : intermediate ; m : mature

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Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and/or IMMP2L. int: intermediate; m: mature form


Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and / or IMMP2L . int : intermediate ; m : mature form . Cells were treated with 5 μM MG132 for 4 h before

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

Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and/or IMMP2L. int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of control or IMMP1L - silenced HeLa cell mitochondria following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers


Western blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of control or IMMP1L - silenced HeLa cell mitochondria transfected with HA - tagged MICU1 WT following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markers . pre : precursor ; int : intermediate ; m : mature form . Cells were treated with 5 μM MG132 for 4 h before

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

Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria transfected with HA-tagged MICU1 WT following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. pre: precursor; int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis


Control or IMMP1L - silenced HeLa cells were transfected with HA - tagged MICU1 WT . Cells were incubated with 50 μg / ml cycloheximide ( CHX ) for the indicated times , collected and subjected to western blotting analysis as indicated . In the graph , the amount of HA ( MICU1 ) is represented relative to the amount at time 0 . ( n = 3 independent experiments )

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Control or IMMP1L-silenced HeLa cells were transfected with HA-tagged MICU1 WT. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1) is represented relative to the amount at time 0. (n=3 independent experiments)


Representative kinetics ( F ) and analysis ( G ) of aequorin - based [ Ca2 + ] m measurements in control or IMMP1L - silenced HeLa cells challenged with 10 μM cyclopiazonic acid ( CPA ) in the presence of 100 μM EGTA ( n = 3 independent experiments )

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Representative kinetics (F) and analysis (G) of aequorin-based [Ca2+]m measurements in control or IMMP1L-silenced HeLa cells challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)


Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in control or IMMP1L - silenced HeLa cells ( n = 3 independent experiments ; 37 - 55 cells ) .

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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in control or IMMP1L-silenced HeLa cells (n=3 independent experiments; 37-55 cells).


Western blot analysis of MICU1 and MICU2 levels in lung cancer cells with low or high Akt activity


Lung cancer cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS ( phospho - Akt substrate ) antibody by western

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

Lung cancer cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS (phospho-Akt substrate) antibody by western blotting


Western blot analysis of MICU1 and MICU2 levels in melanoma cells with low or high Akt activity


Melanoma cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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Melanoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Western blot analysis of MICU1 and MICU2 levels in glioblastoma cells with low or high Akt activity


Glioblastoma cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western

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Glioblastoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting


Number of soft agar colonies ( n = 3 independent experiments formed by Rat2 cells stably expressing the indicated

Number of soft agar colonies (n=3 independent experiments formed by Rat2 cells stably expressing the indicated constructs


xenograft tumor volumes ( H ) and tumor weights at day 21 ( n = 8 mice for each group ) ( I ) formed by Rat2 cells stably expressing the indicated

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xenograft tumor volumes (H) and tumor weights at day 21 (n=8 mice for each group) (I) formed by Rat2 cells stably expressing the indicated constructs


Representative images ( J ) and analysis ( K ) of MitoSOX - based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D ( SD ) or MICU1 S124A ( SA ) ( n = 3 independent experiments ) . Scale bar 10 μm . f . a . u . : fluorescence arbitrary

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

Representative images (J) and analysis (K) of MitoSOX-based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) (n=3 independent experiments). Scale bar 10 μm. f.a.u.: fluorescence arbitrary unit


ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D ( SD ) or MICU1 S124A ( SA ) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated . l . e . : long exposure .

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ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated. l. e.: long exposure.


Isolated sporozoites from the hemolymph ( HL ) and salivary glands ( SG ) of infected mosquitoes stained with Hoechst and SiR - tubulin . Note the similar staining of WT and α1 - tubulin ( - ) complemented lines . Scale bars : 5 µm .

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Isolated sporozoites from the hemolymph (HL) and salivary glands (SG) of infected mosquitoes stained with Hoechst and SiR-tubulin. Note the similar staining of WT and α1-tubulin(-) complemented lines. Scale bars: 5 µm.


SiR - tubulin staining ( green , I and II ) and TEM ( III ) show that microtubules shrink after wild type sporozoite formation . During budding microtubules can be up to 15 µm long ( I ) , while after formation they measure on average only 6 µm ( graph ) . Inset in II shows sporozoite cross - section at the sporoblast membrane ; note the SiR - tubulin fluorescence ( green , arrowheads ) next to the nuclear Hoechst stain ( blue ) . ( III ) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule ( white line , length ) . Green arrowheads point to microtubules close to the sporoblast . Rh : rhoptry ; N : nucleus ; ER : endoplasmic reticulum ; Mi : mitochondrion ; Sb : sporoblast . * * * * indicate p < 0 . 0001 ; Kruskal - Wallis - test . Scale bars : I & II : 5 µm , magnification box in II : 1 µm , III : 1 µm , magnification box in III : 0 . 1 µm .

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SiR-tubulin staining (green, I and II) and TEM (III) show that microtubules shrink after wild type sporozoite formation. During budding microtubules can be up to 15 µm long (I), while after formation they measure on average only 6 µm (graph). Inset in II shows sporozoite cross-section at the sporoblast membrane; note the SiR-tubulin fluorescence (green, arrowheads) next to the nuclear Hoechst stain (blue). (III) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule (white line, length). Green arrowheads point to microtubules close to the sporoblast. Rh: rhoptry; N: nucleus; ER: endoplasmic reticulum; Mi: mitochondrion; Sb: sporoblast. **** indicate p<0.0001; Kruskal-Wallis-test. Scale bars: I&II: 5 µm, magnification box in II: 1 µm, III: 1 µm, magnification box in III: 0.1 µm.


Comparison of sporozoite development in oocysts of WT , α1 - tubulin ( - ) and complemented lines . Hoechst ( blue ) and SiR - tubulin ( green ) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls , while SiR tubulin staining is absent from α1 - tubulin ( - ) oocysts . mCherry stains the cytoplasm of the parasite . Scale bars : 5 µm .

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Comparison of sporozoite development in oocysts of WT, α1-tubulin(-) and complemented lines. Hoechst (blue) and SiR-tubulin (green) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls, while SiR tubulin staining is absent from α1-tubulin(-) oocysts. mCherry stains the cytoplasm of the parasite. Scale bars: 5 µm.


Transmission electron micrographs of developing sporozoites in WT and α1 - tubulin ( - ) parasite lines . Note the slender sporozoites in the WT oocysts , which are absent in the mutant . Sb : sporoblast ; scale bars : 5 µm .

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Transmission electron micrographs of developing sporozoites in WT and α1-tubulin(-) parasite lines. Note the slender sporozoites in the WT oocysts, which are absent in the mutant. Sb: sporoblast; scale bars: 5 µm.


TEM cross sections of WT and α1 - tubulin ( - ) parasites showing the aberrant structure of the latter . Subpellicular and centriolar plaque ( spindle pole body ) associated ( hemispindle ) microtubules are readily recognizable in the WT sporozoites ( green arrowheads in insert ) but absent in the mutant , while the inner membrane complex is formed below the plasma membrane of both parasites ( white arrowheads ) . Note the different scale chosen to highlight the malformed shapes in the mutant . N : nucleus ; scale bars : 0 . 2 µm , magnification boxes : 0 . 1 µm , .

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TEM cross sections of WT and α1-tubulin(-) parasites showing the aberrant structure of the latter. Subpellicular and centriolar plaque (spindle pole body) associated (hemispindle) microtubules are readily recognizable in the WT sporozoites (green arrowheads in insert) but absent in the mutant, while the inner membrane complex is formed below the plasma membrane of both parasites (white arrowheads). Note the different scale chosen to highlight the malformed shapes in the mutant. N: nucleus; scale bars: 0.2 µm, magnification boxes: 0.1 µm,.


Electron micrographs showing different stages of development in WT and α1 - tubulin ( - ) parasites . Key structural elements are highlighted in the following colors : blue : plasma membrane ; yellow : inner membrane complex ; green : microtubules ; light blue : micronemes ; lilac : rhoptries ; brown : rootlet fiber ; cyan : nucleus . Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1 - tubulin ( - ) parasites . The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque ( dark structure in nuclear envelope ) . Panel III corresponds to Fig 1D . Scale bars : 200 nm .

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Electron micrographs showing different stages of development in WT and α1-tubulin(-) parasites. Key structural elements are highlighted in the following colors: blue: plasma membrane; yellow: inner membrane complex; green: microtubules; light blue: micronemes; lilac: rhoptries; brown: rootlet fiber; cyan: nucleus. Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1-tubulin(-) parasites. The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque (dark structure in nuclear envelope). Panel III corresponds to Fig 1D. Scale bars: 200 nm.


α1 - tubulin ( - ) parasites show multiple attachments ( arrowheads ) with the sporoblast membrane at day 17 post infection . Note the highly branched nature of the budding parasite ( same parasite as in panel C ) shown in two views in I and II . Right : slices through the tomogram in grey scale with the outlines used for the 3D model indicated . Two connections with the sporoblast are indicated by 1 and 2 . Scale bars : 1 µm .

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α1-tubulin(-) parasites show multiple attachments (arrowheads) with the sporoblast membrane at day 17 post infection. Note the highly branched nature of the budding parasite (same parasite as in panel C) shown in two views in I and II. Right: slices through the tomogram in grey scale with the outlines used for the 3D model indicated. Two connections with the sporoblast are indicated by 1 and 2. Scale bars: 1 µm.


Life imaging of wild - type ( WT ) and α1 - tubulin ( - ) oocysts expressing ef1α : GFP ( early and late oocysts ) and csp : mCherry ( late oocysts ) to locate the oocyst cytoplasm . Microtubules were labeled with SiR - tubulin ( green ) and DNA with Hoechst ( blue ) . Oocysts are shown in a chronological order from early ( day 4 ) to late ( day 12 ) development . ( A , B ) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage ( A ) and subsequent DNA replication ( B ) , where some but not all DNA is co - stained by microtubules in the mutant . ( C ) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites . Note the strong SiR - tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant . Sb : sporoblast . ( D ) Oocyst ready to burst with fully formed sporozoites in WT oocysts . Note that α1 - tubulin ( - ) oocysts retained sporozoite nuclei predominantly within the sporoblast ( Sb ) during budding . Scale bars : 5 μm , magnification box in B : 1 µm , .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O 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-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 O O O O O O O O O O O O O O O O O O

Life imaging of wild-type (WT) and α1-tubulin(-) oocysts expressing ef1α:GFP (early and late oocysts) and csp:mCherry (late oocysts) to locate the oocyst cytoplasm. Microtubules were labeled with SiR-tubulin (green) and DNA with Hoechst (blue). Oocysts are shown in a chronological order from early (day 4) to late (day 12) development. (A, B) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage (A) and subsequent DNA replication (B), where some but not all DNA is co-stained by microtubules in the mutant. (C) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites. Note the strong SiR-tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant. Sb: sporoblast. (D) Oocyst ready to burst with fully formed sporozoites in WT oocysts. Note that α1-tubulin(-) oocysts retained sporozoite nuclei predominantly within the sporoblast (Sb) during budding. Scale bars: 5 μm, magnification box in B: 1 µm,.


3D reconstructions from spinning disc confocal microscopy z - stacks across an early oocyst labelled with the DNA dye Hoechst ( left ) . 50 and 19 images with a z - distance of 0 . 5 µm and 1 µm were collated for WT and α1 - tubulin ( - ) oocysts , respectively . Right : 3D reconstructions from late WT and α1 - tubulin ( - ) oocysts labelled with Hoechst ( blue ) and SiR - tubulin ( green ) . Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites . 10 and 28 images with a z - distance of 1 , 5 µm and 1 µm were collated for WT and α1 - tubulin ( - ) oocysts , respectively . Sb : sporoblast ; scale bars : 5 µm .

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

3D reconstructions from spinning disc confocal microscopy z-stacks across an early oocyst labelled with the DNA dye Hoechst (left). 50 and 19 images with a z-distance of 0.5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Right: 3D reconstructions from late WT and α1-tubulin(-) oocysts labelled with Hoechst (blue) and SiR-tubulin (green). Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites. 10 and 28 images with a z-distance of 1,5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Sb: sporoblast; scale bars: 5 µm.


TEM cross sections of WT and α1 - tubulin ( - ) oocysts during nuclear division . Spindle microtubules ( arrowheads ) emerging from centriolar plaques ( M ) were seen in WT oocysts . Although individual nuclei ( N ) were visible in α1 ( - ) oocysts , microtubules were absent even in dividing centriolar plaques ( M ) . Scale bars : 0 . 2 μm .

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TEM cross sections of WT and α1-tubulin(-) oocysts during nuclear division. Spindle microtubules (arrowheads) emerging from centriolar plaques (M) were seen in WT oocysts. Although individual nuclei (N) were visible in α1(-) oocysts, microtubules were absent even in dividing centriolar plaques (M). Scale bars: 0.2 μm.


Fluorescence images showing the SiR - tubulin stained microtubules , nuclei ( Hoechst ) and cytoplasmic shape ( mCherry ) of the parasite lines . Note the small amount of DNA ( black arrowhead ) and the reduced level of SiR - tubulin fluorescence in the α1cm & ∆ introns - expressing sporozoites ( lower graph ) . See also Fig S6 . Quantitative data was derived from 96 - 120 sporozoites per line . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . Scale bar : 5 μm . * * * * indicates p < 0 . 0001 , Kruskal - Wallis - test . Note that α1cm & ∆ introns - expressing sporozoites do not express mCherry ; their autofluorescence ( auto - fluo ) indicates an aberrant shape .

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

Fluorescence images showing the SiR-tubulin stained microtubules, nuclei (Hoechst) and cytoplasmic shape (mCherry) of the parasite lines. Note the small amount of DNA (black arrowhead) and the reduced level of SiR-tubulin fluorescence in the α1cm&∆introns-expressing sporozoites (lower graph). See also Fig S6. Quantitative data was derived from 96-120 sporozoites per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. Scale bar: 5 μm. **** indicates p<0.0001, Kruskal-Wallis-test. Note that α1cm&∆introns-expressing sporozoites do not express mCherry; their autofluorescence (auto-fluo) indicates an aberrant shape.