Figure 2 ( A ) Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy to light ( H / L ) and ( L / H ) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and ∆ 4 - CYB cells The logarithmic ratios were calculated using MaxQuant and the statistical significance of the differences for the enrichment or depletion of the proteins were determined with Perseus ( B ) Labeling of the thirteen mtDNA - encoded MRC structural subunits . Cells were incubated with [ 35S ] - L - Met for one hour in the presence of emetine 100 μg / ml to inhibit cytoplasmic translation . ( C ) Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 ; * * * p = 0 . 0007 . ( D ) Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 ; * * p = 0 . 0024 ( NDUFS3 ) ; * * p = 0 . 0061 ( NDUFB8 ) . ( E ) Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0011 . ( F ) Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 . ( G ) Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . There were no differences in the steady - state levels of the tested subunits ( 2 - way ANOVA with Sidak ' s multiple comparisons test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy to light (H/L) and (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and ∆4-CYB cells The logarithmic ratios were calculated using MaxQuant and the statistical significance of the differences for the enrichment or depletion of the proteins were determined with Perseus(B) Labeling of the thirteen mtDNA-encoded MRC structural subunits. Cells were incubated with [35S]-L-Met for one hour in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation.(C) Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001; ***p=0.0007.(D) Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001; **p=0.0024 (NDUFS3); **p=0.0061 (NDUFB8).(E) Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0011.(F) Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001.(G) Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). There were no differences in the steady-state levels of the tested subunits (2-way ANOVA with Sidak's multiple comparisons test).


Figure 3 ( A ) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised The graphs plot the relative peptide peak intensities along the lane , setting the maximum to 1 . 0 , vs . the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve . The relative amounts of the proteins between the two cell lines was determined by calculating the H / L ratios of peptides that were present in both WT ( blue traces ) and ∆ 4 - CYB samples ( red traces ) . The represented values are the mean ± SEM of the two reciprocal labeling experiments . ( B ) Second - dimension BNGE of digitonin - solubilized samples from WT and ∆ 4 - CYB cells , western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies . The immunodetection patterns were equivalent to the complexome profiles . ( C ) Quantification of the total peak area under the curves ( AUC ) defined by the peptide intensity peaks for the indicated cIII2 subunits . The x - axis values were the slice number ( 1 - 64 ) and the y - axis values were the relative peptide intensity . The graph shows the mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0083 ( UQCRC2 ) ; * * p = 0 . 0033 ( UQCRFS1 ) ; * p = 0 . 0224 ; n . s . = non - significant . ( D ) Complexome profiles of two cIII2 assembly factors ( BCS1L and LYRM7 or MZM1L ) generated The represented values are the mean ± SEM of the two reciprocal labeling experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, vs. the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines was determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and ∆4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments.(B) Second-dimension BNGE of digitonin-solubilized samples from WT and ∆4-CYB cells, western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles.(C) Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII2 subunits. The x-axis values were the slice number (1-64) and the y-axis values were the relative peptide intensity. The graph shows the mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0083 (UQCRC2); **p=0.0033 (UQCRFS1); *p=0.0224; n.s.=non-significant.(D) Complexome profiles of two cIII2 assembly factors (BCS1L and LYRM7 or MZM1L) generated The represented values are the mean ± SEM of the two reciprocal labeling experiments.


Figure 4 ( A ) SDS - PAGE , Western blot an immunodetection , with the indicated specific antibodies , of ∆ 4 - CYB and WT cells expressing HA - tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert ( Empty ) . ( B ) BNGE , Western blot and immunodetection , with an anti - HA tag antibody , of samples from the same cell lines as in ( A ) solubilized either with digitonin or DDM . ( C ) BNGE , Western blot and immunodetection , with the monoclonal ( M ) anti - UQCRQ antibody ( Abcam ab110255 ) , of non - transduced ∆ 4 - CYB and WT cells . The mitoplast samples were solubilized with DDM ( D ) Scatter plot generated from the analysis of the logarithmic heavy ( H ) to light ( L ) ratios in the x - axis and the reverse in the y - axis , in the two reciprocal labeling SILAC experiments ( 1 and 2 ) and anti - HA immunopurification of ∆ 4 - CYB and WT cells expressing UQCR10HA . ( E ) Complexome profiles for the proteins found specifically enriched in ∆ 4 - CYB UQCR10HA , according to the SILAC immunopurification experiments The represented values are the mean ± SEM of the two reciprocal labeling experiments . ( F ) SDS - PAGE , Western blot and immunodetection , with the indicated specific antibodies , of ∆ 4 - CYB and WT cells expressing an HA - tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert ( Empty ) . ( G ) BNGE , Western blot and immunodetection , with an anti - HA tag antibody , of samples from the cell lines solubilized either with digitonin . ( H ) Scatter plot generated from the analysis of the logarithmic heavy ( H ) to light ( L ) ratios in the x - axis and the reverse in the y - axis , in the two reciprocal labeling SILAC experiments of anti - HA immunopurification of ∆ 4 - CYB and WT cells expressing CYC1HA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) SDS-PAGE, Western blot an immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing HA-tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).(B) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM.(C) BNGE, Western blot and immunodetection, with the monoclonal (M) anti-UQCRQ antibody (Abcam ab110255), of non-transduced ∆4-CYB and WT cells. The mitoplast samples were solubilized with DDM(D) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments (1 and 2) and anti-HA immunopurification of ∆4-CYB and WT cells expressing UQCR10HA.(E) Complexome profiles for the proteins found specifically enriched in ∆4-CYB UQCR10HA, according to the SILAC immunopurification experiments The represented values are the mean ± SEM of the two reciprocal labeling experiments.(F) SDS-PAGE, Western blot and immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing an HA-tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).(G) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the cell lines solubilized either with digitonin.(H) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments of anti-HA immunopurification of ∆4-CYB and WT cells expressing CYC1HA.


Figure 5 ( A ) Complexome profiles of the different cI structural modules . The graphs were generated but in this case , the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis The represented values are the mean ± SEM of the two reciprocal labeling experiments . ( B ) Complexome profiles of the cI assembly factor NDUFAF2 in WT ( blue ) and ∆ 4 - CYB ( red ) mitochondria The represented values are the mean ± SEM of the two reciprocal labeling experiments . ( C ) Quantification of the total area under the curve in the profiles corresponding to each cI module ( top graph ) . The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules . The plotted values are the mean mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * p = 0 . 0006 ; * * p = 0 . 0012 ( N - module ) ; * * p = 0 . 0016 ( ND1 - module ) ; * * p = 0 . 0032 ( ND2 - module ) ; * * p = 0 . 0057 ( ND4 - module ) ; * * p = 0 . 0020 ( ND5 - module ) ; * p = 0 . 0140 . ( D ) Quantification of the area of the N - module peak corresponding to the molecular mass of the fully assembled cI : 1 , 172 kDa in ∆ 4 - CYB samples and the respirasome ( cI + cIII2 + cIV ) SCs in # 4 . 1 WT samples ( 2 , 110 kDa ) . The plotted values are the mean mean ± SD ( n = 2 ) . Unpaired Student ' s t - test * * * * p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Complexome profiles of the different cI structural modules. The graphs were generated but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis The represented values are the mean ± SEM of the two reciprocal labeling experiments.(B) Complexome profiles of the cI assembly factor NDUFAF2 in WT (blue) and ∆4-CYB (red) mitochondria The represented values are the mean ± SEM of the two reciprocal labeling experiments.(C) Quantification of the total area under the curve in the profiles corresponding to each cI module (top graph). The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules. The plotted values are the mean mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test ***p=0.0006; **p=0.0012 (N-module); **p=0.0016 (ND1-module); **p=0.0032 (ND2-module); **p=0.0057 (ND4-module); **p=0.0020 (ND5-module); *p=0.0140.(D) Quantification of the area of the N-module peak corresponding to the molecular mass of the fully assembled cI: 1,172 kDa in ∆4-CYB samples and the respirasome (cI+cIII2+cIV) SCs in #4.1 WT samples (2,110 kDa). The plotted values are the mean mean ± SD (n=2). Unpaired Student's t-test ****p<0.0001.


Figure 6 ( A ) SDS - PAGE resolving the radioactively labeled mitochondrial translation products after a 2 - hour pulse ( P ) . The 35S - Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times ( 2 , 5 and 24 hours ) . ( B ) First dimension ( 1D ) BNGE analysis of the same cells prepared with digitonin . ( C ) Denaturing second dimension ( 2D ) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species . ( D ) Complex I IGA ( cI - IGA ) analysis of digitonin - solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days ( 0h ) . After removing the drug and restoring synthesis of the mtDNA - encoded subunits , the cells were collected at the indicated times to follow the appearance of cI reactivity with time . The gels were incubated in the reaction mixture for 24 hours . SS = steady state . The asterisk indicates the presence of a high molecular weight cI - containing band of unknown nature ( E ) 2D BNGE , Western blot and immunodetection analysis of WT and ∆ 4 - CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear - encoded subunits , belonging to different structural modules . The blots shown were either exposed for 16 sec . ( low exposures ) or 160 sec . ( high exposures ) in order to visualize the qualitative signals in the ∆ 4 - CYB samples .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) SDS-PAGE resolving the radioactively labeled mitochondrial translation products after a 2-hour pulse (P). The 35S-Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times (2, 5 and 24 hours).(B) First dimension (1D) BNGE analysis of the same cells prepared with digitonin.(C) Denaturing second dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species.(D) Complex I IGA (cI-IGA) analysis of digitonin-solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0h). After removing the drug and restoring synthesis of the mtDNA-encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 hours. SS = steady state. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature(E) 2D BNGE, Western blot and immunodetection analysis of WT and ∆4-CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear-encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 sec. (low exposures) or 160 sec. (high exposures) in order to visualize the qualitative signals in the ∆4-CYB samples.


Figure 7 ( A ) SDS - PAGE , Western blot and immunodetection of AOXHA expression in whole - cell lysates from WT and ∆ 4 - CYB cells transduced with the AOXHA / pWPXLd - ires - HygroR lentiviral vector . The transduction and selection controls were the same cell lines transfected with an empty pWPXLd - ires - HygroR vector ( EV ) . ( B ) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls . Cell growth was monitored every six hours after substituting the medium in two replicate 24 - well plates , one plate with medium without uridine ( Uridine - ) , and the second plate with medium supplemented with 50 µg / ml uridine ( Uridine + ) . The graphs show the average confluence ± SD at each time point ( n = 6 wells per cell line ) . ( C ) Immunodetection of cI structural subunits and NDUFAF2 in the same samples ( D ) 1D BNGE , Western blot and immunodetection analyses of digitonin - solubilized mitochondria from WT and ∆ 4 - CYB cybrids expressing AOXHA and their corresponding EV controls . ( E ) Complex I in gel activity assays ( IGA ) after BNGE The gels were incubated in the IGA reaction mixture for 5 hours . The asterisk indicates the presence of a high molecular weight cI - containing band of unknown nature ( F ) Spectrophotometric kinetic measurements of cI activity in WT and ∆ 4 - CYB AOXHA and EV samples , normalized by the percentage of citrate synthase ( CS ) activity . Results are expressed as mean ± SD ( n = 5 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * p = 0 . 0077 ( WT AOXHA vs . ∆ 4 - CYB AOXHA ) ; * * p = 0 . 0044 ( ∆ 4 - CYB EV vs . ∆ 4 - CYB AOXHA ) ; * * * p = 0 . 0003 ; * * * * p < 0 . 0001 . ( G ) High resolution respirometry analyses performed in intact cells in an Oroboros instrument . ROUTINE : cellular basal oxygen consumption rate ( in pmol O2 / sec ) per million cells in DMEM medium . LEAK is the non - phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin . ETS : Maximal respiration rate in the presence of the uncoupler CCCP . AOX : oxygen consumption in the presence of antimycin A , inhibiting cIII2 activity but not that of AOX . ROTENONE : Oxygen consumption in the presence of the cI inhibitor rotenone . In all cases this was equal to the background . Results are expressed as mean ± SD ( n = 2 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * * * p < 0 . 0001 . ( H ) Respirometry analyses , in WT EV controls and AOXHA - expressing cells untreated or treated with 2 . 5 µM antimycin A for 7 days ( + AA ) . Results are expressed as mean ± SD ( n = 4 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * * * p < 0 . 0001 . ( I ) 1D BNGE followed by cI - IGA ( right ) or Western blot and immunodetection of cIII2 subunit CYC1 ( left ) in digitonin - solubilized samples from WT EV controls and AOXHA - expressing cells untreated ( - ) or treated ( + ) with 2 . 5 µM antimycin A for 7 days .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) SDS-PAGE, Western blot and immunodetection of AOXHA expression in whole-cell lysates from WT and ∆4-CYB cells transduced with the AOXHA/pWPXLd-ires-HygroR lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd-ires-HygroR vector (EV).(B) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls. Cell growth was monitored every six hours after substituting the medium in two replicate 24-well plates, one plate with medium without uridine (Uridine-), and the second plate with medium supplemented with 50 µg/ml uridine (Uridine+). The graphs show the average confluence ± SD at each time point (n=6 wells per cell line).(C) Immunodetection of cI structural subunits and NDUFAF2 in the same samples(D) 1D BNGE, Western blot and immunodetection analyses of digitonin-solubilized mitochondria from WT and ∆4-CYB cybrids expressing AOXHA and their corresponding EV controls.(E) Complex I in gel activity assays (IGA) after BNGE The gels were incubated in the IGA reaction mixture for 5 hours. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature(F) Spectrophotometric kinetic measurements of cI activity in WT and ∆4-CYB AOXHA and EV samples, normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=5 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test **p=0.0077 (WT AOXHA vs. ∆4-CYB AOXHA); **p=0.0044 (∆4-CYB EV vs. ∆4-CYB AOXHA); ***p=0.0003; ****p<0.0001.(G) High resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non-phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: Maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: Oxygen consumption in the presence of the cI inhibitor rotenone. In all cases this was equal to the background. Results are expressed as mean ± SD (n=2 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.(H) Respirometry analyses , in WT EV controls and AOXHA- expressing cells untreated or treated with 2.5 µM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n=4 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.(I) 1D BNGE followed by cI-IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin-solubilized samples from WT EV controls and AOXHA- expressing cells untreated (-) or treated (+) with 2.5 µM antimycin A for 7 days.


Figure 8 ( A ) Complexome profiles of the two cV structural and assembly modules . The graphs were generated as in Figure 3 but in this case , the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis . The represented values are the mean ± SEM of the two reciprocal labeling experiments . ( B ) Complexome profiles of the three detected cII subunits . The represented values are the mean ± SEM of the two reciprocal labeling experiments . ( C ) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated ( NDUFA4 ) . , the peptide intensity values for the individual subunits belonging to each assembly module were averaged to simplify the analysis . The represented values are the mean ± SEM of the two reciprocal labeling experiments . The bar graph represents the quantification of the total area under the curve in the profiles corresponding to each cIV module . The plotted values are mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0088 ( Early ) ; * * p = 0 . 0063 ( MT - CO2 ) ; * * p = 0 . 0040 ( NDUFA4 ) ; * p = 0 . 0264 . ( D ) 1D BNGE , Western blot and immunodetection of two cIV subunits ( MT - CO1 and COX7B ) in samples from WT and ∆ 4 - CYB cells solubilized with DDM and digitonin ( Dig ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Figure 3 but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments.(B) Complexome profiles of the three detected cII subunits. The represented values are the mean ± SEM of the two reciprocal labeling experiments.(C) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). , the peptide intensity values for the individual subunits belonging to each assembly module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve in the profiles corresponding to each cIV module. The plotted values are mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0088 (Early); **p=0.0063 (MT-CO2); **p=0.0040 (NDUFA4); *p=0.0264.(D) 1D BNGE, Western blot and immunodetection of two cIV subunits (MT-CO1 and COX7B) in samples from WT and ∆4-CYB cells solubilized with DDM and digitonin (Dig).


Figure 1 ( A , B ) qRT - PCR analysis of IL6 expression upon indicated stimulations in presence or absence of NOD1 ( A ) or NOD2 ( B ) . HeLa inducible NOD1 cells ( A ) or NOD2 cells ( B ) were induced in absence or presence of doxycycline overnight and afterwards stimulated with various stimuli for 4 h .

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Figure 1(A, B) qRT-PCR analysis of IL6 expression upon indicated stimulations in presence or absence of NOD1 (A) or NOD2 (B). HeLa inducible NOD1 cells (A) or NOD2 cells (B) were induced in absence or presence of doxycycline overnight and afterwards stimulated with various stimuli for 4 h.


Figure 2 ( C ) qRT - PCR analysis of Cxcl2 expression upon indicated stimulations in WT , Nod1 / 2 KO and Rip2 KO bone marrow derived macrophages ( BMDMs ) .

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Figure 2(C) qRT-PCR analysis of Cxcl2 expression upon indicated stimulations in WT, Nod1/2 KO and Rip2 KO bone marrow derived macrophages (BMDMs).


Figure 3ELISA analysis of IL6 in supernatants of HDFs ( E ) upon indicated stimulations for 20 h in absence or presence of the inhibitor of sphingosine kinases . ( H ) qRT - PCR analysis of Cxcl2 expression in WT , Sphk1 KO or Sphk2 KO BMDMs upon indicated stimulations for 4 h .

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Figure 3ELISA analysis of IL6 in supernatants of HDFs (E) upon indicated stimulations for 20 h in absence or presence of the inhibitor of sphingosine kinases.(H) qRT-PCR analysis of Cxcl2 expression in WT, Sphk1 KO or Sphk2 KO BMDMs upon indicated stimulations for 4 h.


Figure 4 ( D ) Western blot analysis of MAPKs and NF - κB activation upon S1P stimulation . HeLa NOD1 or NOD2 cells were treated with cytosolic S1P ( 20 µM ) , iE - DAP ( 20 µM ) or MDP ( 20 µM ) for indicated times . Multiplex analysis of CXCL2 ( I ) , CCL5 ( J ) and IL16 ( K ) production in supernatants of WT , Nod1 / 2 and Rip2 KO BMDMs upon S1P ( 10 µM ) or MDP ( 10 µM ) stimulations together with digitonin ( 2 . 5 µg / ml ) for 20 h .

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Figure 4(D) Western blot analysis of MAPKs and NF-κB activation upon S1P stimulation. HeLa NOD1 or NOD2 cells were treated with cytosolic S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) for indicated times.Multiplex analysis of CXCL2 (I), CCL5 (J) and IL16 (K) production in supernatants of WT, Nod1/2 and Rip2 KO BMDMs upon S1P (10 µM) or MDP (10 µM) stimulations together with digitonin (2.5 µg/ml) for 20 h.


Figure 5 ( A , B ) Immunoprecipitation with various lipid - coated beads using HeLa NOD1 - GFP ( A ) and NOD2 - GFP cells ( B ) . Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid - coated beads at room temperature for 2 h . ( E , F ) Single particle tracking of NOD1 - GFP ( E ) or NOD2 - GFP ( F ) with S1P - TAMRA . After doxycycline induction overnight , HeLa NOD1 or NOD2 cells were stimulated with S1P - TAMRA ( 20 µM ) for 1 h together with digitonin . Scale bar : 5 µm .

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Figure 5(A, B) Immunoprecipitation with various lipid-coated beads using HeLa NOD1-GFP (A) and NOD2-GFP cells (B). Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid-coated beads at room temperature for 2 h.(E, F) Single particle tracking of NOD1-GFP (E) or NOD2-GFP (F) with S1P-TAMRA. After doxycycline induction overnight, HeLa NOD1 or NOD2 cells were stimulated with S1P-TAMRA (20 µM) for 1 h together with digitonin. Scale bar: 5 µm.


Figure 6 ( D ) MST analysis of direct binding of NOD1 to S1P and iE - DAP . ( E ) MST analysis of direct binding of NOD2 with S1P and MDP . ( F ) ELISA analysis of IL8 in supernatants of HEK293T NOD1 / 2 KO cells transfected with indicated NOD1 or NOD2 mutants . After 24 h transfection , cells were stimulated with S1P ( 20 µM ) , iE - DAP ( 20 µM ) or MDP ( 20 µM ) together with digitonin for 20 h .

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Figure 6(D) MST analysis of direct binding of NOD1 to S1P and iE-DAP. (E) MST analysis of direct binding of NOD2 with S1P and MDP.(F) ELISA analysis of IL8 in supernatants of HEK293T NOD1/2 KO cells transfected with indicated NOD1 or NOD2 mutants. After 24 h transfection, cells were stimulated with S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) together with digitonin for 20 h.


Figure 7 ( A , B ) MST analysis of direct binding of NOD1 ( A ) or NOD2 ( B ) with ADP in the presence of S1P ( 2 µM ) .

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Figure 7(A, B) MST analysis of direct binding of NOD1 (A) or NOD2 (B) with ADP in the presence of S1P (2 µM).


Figure 1A - O ( A , F , K ) slices through electron cryo - tomograms of L . pneumophila , P . aeruginosa , and S . oneidensis cells , respectively , highlighting a PL sub - complex in the outer membrane ( dashed yellow circle ) . ( B , G , L ) Enlarged views of the complexes . ( C , H , M ) Sub - tomogram averages of PL sub - complexes from each species . ( D , I , N ) Schematic representations of the sub - tomogram averages with different rings colored and labeled . ( E , J , O ) Sub - tomogram averages of fully - assembled flagella from each species for comparison . Scale bars : ( black ) 50 nm , ( orange ) 25 nm , ( red ) 20 nm .

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Figure 1A-O (A, F, K) slices through electron cryo-tomograms of L. pneumophila, P. aeruginosa, and S. oneidensis cells, respectively, highlighting a PL sub-complex in the outer membrane (dashed yellow circle). (B, G, L) Enlarged views of the complexes. (C, H, M) Sub-tomogram averages of PL sub-complexes from each species. (D, I, N) Schematic representations of the sub-tomogram averages with different rings colored and labeled. (E, J, O) Sub-tomogram averages of fully-assembled flagella from each species for comparison. Scale bars: (black) 50 nm, (orange) 25 nm, (red) 20 nm.


Figure 2Sub - tomogram average of the IM sub - complex constituting the C - ring , MS - ring and export apparatus . Schematic representation of the sub - tomogram average shown in ( A ) highlighting the different parts of the complex . Sub - tomogram average of the motors of fully - assembled flagella . Slices through electron cryo - tomograms showing neighboring PL and IM sub - complexes . Dashed - yellow circles highlight the IM sub - complex while dashed - blue arrows highlight the PL sub - complex . Dashed - red lines mark the border between two images used to make a composite image when the PL and IM sub - complexes were found at different levels in the tomogram . Schematic representation of an IM sub - complex in the vicinity of a PL sub - complex . Slices through electron cryo - tomograms of L . pneumophila highlighting the presence of a fully - assembled basal body lacking the hook and the filament . Schematic representation of the basal bodies in ( L and M ) . Central slice through an electron cryo - tomogram of a lysed cell . The dashed - yellow circle highlights the flagellar motor . Enlarged view of the same slice shown in ( O ) . The absence of the C - ring and the export apparatus is highlighted by the dashed - orange arrow . Schematic representation of the complex found in ( P ) .

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Figure 2Sub-tomogram average of the IM sub-complex constituting the C- ring, MS-ring and export apparatus. Schematic representation of the sub-tomogram average shown in (A) highlighting the different parts of the complex. Sub-tomogram average of the motors of fully-assembled flagella. Slices through electron cryo-tomograms showing neighboring PL and IM sub-complexes. Dashed-yellow circles highlight the IM sub-complex while dashed-blue arrows highlight the PL sub-complex. Dashed-red lines mark the border between two images used to make a composite image when the PL and IM sub-complexes were found at different levels in the tomogram. Schematic representation of an IM sub-complex in the vicinity of a PL sub-complex. Slices through electron cryo-tomograms of L. pneumophila highlighting the presence of a fully-assembled basal body lacking the hook and the filament. Schematic representation of the basal bodies in (L and M). Central slice through an electron cryo-tomogram of a lysed cell. The dashed-yellow circle highlights the flagellar motor. Enlarged view of the same slice shown in (O). The absence of the C-ring and the export apparatus is highlighted by the dashed-orange arrow. Schematic representation of the complex found in (P).


Figure 3Slices through electron cryo - tomograms showing fully - assembled motors without the hook and filament . The dashed - yellow circles indicate the IM sub - complex . Schematic representation of the P . aeruginosa motors lacking the hook and filament shown in ( A - C ) . Slices through electron cryo - tomograms showing fully - assembled motors with the hook and lacking the filament . Schematic representation of the motors with the hook shown in ( E - G ) . Slices through electron cryo - tomograms of intact P . aeruginosa ∆ flgI cells showing the presence of the inner - membrane sub - complex with the rod . Schematic representation of the inner - membrane sub - complex with the rod shown in ( I - K ) . Slices through electron cryo - tomogram of lysed P . aeruginosa ∆ flgI cells showing the presence of the sub - complex constituting the MS - ring and the rod . Schematic representation of the sub - complex described in ( M - O ) . A slice through an electron cryo - tomogram of a P . aeruginosa ∆ flgG cell highlighting the presence of the inner - membrane sub - complex . Schematic representation of the inner - membrane sub - complex shown in ( Q ) .

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Figure 3Slices through electron cryo-tomograms showing fully-assembled motors without the hook and filament. The dashed-yellow circles indicate the IM sub-complex. Schematic representation of the P. aeruginosa motors lacking the hook and filament shown in (A-C). Slices through electron cryo-tomograms showing fully-assembled motors with the hook and lacking the filament. Schematic representation of the motors with the hook shown in (E-G). Slices through electron cryo-tomograms of intact P. aeruginosa ∆flgI cells showing the presence of the inner-membrane sub-complex with the rod. Schematic representation of the inner-membrane sub-complex with the rod shown in (I-K). Slices through electron cryo-tomogram of lysed P. aeruginosa ∆flgI cells showing the presence of the sub-complex constituting the MS-ring and the rod. Schematic representation of the sub-complex described in (M-O). A slice through an electron cryo-tomogram of a P. aeruginosa ∆flgG cell highlighting the presence of the inner-membrane sub-complex. Schematic representation of the inner-membrane sub-complex shown in (Q).


Figure 4Slices through electron cryo - tomograms of wild type cells showing fully - assembled motors without the hook and filament . Schematic representation of the motors lacking the hook and filament shown in ( A - C ) . Sub - tomogram average of the motors of fully - assembled flagella . Slice through an electron cryo - tomogram of a ΔflgH cell showing an IM sub - complex , indicated by the dashed - yellow circle . Schematic representation of the IM sub - complex shown in ( F ) . Slices through electron cryo - tomograms of ΔflgH cells showing the IM sub - complex with the rod and the P - ring , indicated by the dashed - yellow circles . Schematic representation of the structures shown in ( H and I ) . Slices through electron cryo - tomograms of ΔflaA / B cells highlighting the flagellar motor and the hook ( without the filament ) . Sub - tomogram average of the flagellar motor and the hook structure found in ΔflaA / B cells . Schematic representation of the sub - tomogram average shown in ( N ) . Slices through electron cryo - tomograms of ΔflaA / B cells illustrating a disassembly product constituting the PL sub - complex , the rod and the hook . Sub - tomogram average of the disassembly complex shown in ( P - R ) . The dashed - orange arrow indicates the absence of the IM sub - complex in this structure . Schematic representation of the disassembly product shown in ( P - S ) . Slices through electron cryo - tomograms of ΔflaA / B cells highlighting PL sub - complexes ( dashed - yellow circles ) . Sub - tomogram average of the PL sub - complexes in ΔflaA / B cells . Schematic representation of the sub - tomogram average shown in ( X ) .

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Figure 4Slices through electron cryo-tomograms of wild type cells showing fully-assembled motors without the hook and filament. Schematic representation of the motors lacking the hook and filament shown in (A-C). Sub-tomogram average of the motors of fully-assembled flagella. Slice through an electron cryo-tomogram of a ΔflgH cell showing an IM sub-complex, indicated by the dashed-yellow circle. Schematic representation of the IM sub-complex shown in (F). Slices through electron cryo-tomograms of ΔflgH cells showing the IM sub-complex with the rod and the P-ring, indicated by the dashed-yellow circles. Schematic representation of the structures shown in (H and I). Slices through electron cryo-tomograms of ΔflaA/B cells highlighting the flagellar motor and the hook (without the filament). Sub-tomogram average of the flagellar motor and the hook structure found in ΔflaA/B cells. Schematic representation of the sub-tomogram average shown in (N). Slices through electron cryo-tomograms of ΔflaA/B cells illustrating a disassembly product constituting the PL sub-complex, the rod and the hook. Sub-tomogram average of the disassembly complex shown in (P-R). The dashed-orange arrow indicates the absence of the IM sub-complex in this structure. Schematic representation of the disassembly product shown in (P-S). Slices through electron cryo-tomograms of ΔflaA/B cells highlighting PL sub-complexes (dashed-yellow circles). Sub-tomogram average of the PL sub-complexes in ΔflaA/B cells. Schematic representation of the sub-tomogram average shown in (X).


Figure 1C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in control and PHSL1 - CDC20 cells . Top , time - lapse series . Bottom , meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . Data information : data are representative of four independent experiments . D . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 strains containing the indicated mutations . Meiotic events were analyzed as in ( C ) . Data information : data are representative of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in control and PHSL1-CDC20 cells. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of four independent experiments.D. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 strains containing the indicated mutations. Meiotic events were analyzed as in (C). Data information: data are representative of two independent experiments.


Figure 2A . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control cells and spo13 mutants . Top , time - lapse series . Bottom , meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . Data information : data are representative of two independent experiments . C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control cells and cells containing clb1 ∆ or cdc28 - as1 , which have been treated with 1NM - PP1 at metaphase I ( 8 h in SPM ) . Meiotic events ( bottom ) were analyzed as in ( A ) . Data information : data are representative of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and spo13 mutants. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of two independent experiments.C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and cells containing clb1∆ or cdc28-as1, which have been treated with 1NM-PP1 at metaphase I (8 h in SPM). Meiotic events (bottom) were analyzed as in (A). Data information: data are representative of two independent experiments.


Figure 3A . Imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ cells containing SPC72 , spc72 - 7 , or spc72 - 7 plus hrr25 - as . Cells were shifted from 24 to 36 ° C at 4 h in SPM and treated with 1NM - PP1 to inhibit Hrr25 - as . Top , time - lapse series . Bottom , Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Data information : data are representative of three independent experiments . C . Imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ spo13 ∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM . Mpc70 loading ( bottom ) was analyzed as in ( A ) . PEST - SPC72 expression delays the time of Mpc70 loading by 98 min ( 95 % CI , 67 - 129 ; P < 0 . 0001 ; Welch ' s t - test ) . Data information : data are representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus hrr25-as. Cells were shifted from 24 to 36°C at 4 h in SPM and treated with 1NM-PP1 to inhibit Hrr25-as. Top, time-lapse series. Bottom, Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Data information: data are representative of three independent experiments.C. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ spo13∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM. Mpc70 loading (bottom) was analyzed as in (A). PEST-SPC72 expression delays the time of Mpc70 loading by 98 min (95% CI, 67-129; P < 0.0001; Welch's t-test). Data information: data are representative of three independent experiments.


Figure 4 ( B ) Analysis of cells containing wild - type Spo13 , Spo13 - mD , or Spo13 - mD - m2 . Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . spo13 - mD delays Spc72 removal and Mpc70 loading by 30 min ( 95 % CI , 26 - 34 ; P < 0 . 0001 ) and 60 min ( 95 % CI , 55 - 66 ; P < 0 . 0001 ; Welch ' s t - test ) , respectively . ( C ) Analysis of cells expressing Clb1 - mDK and different versions of Spo13 . Meiotic events were quantified as in ( B ) . spo13 - mD plus PEST - clb1 - mDK delays Spc72 removal and Mpc70 loading by 72 min ( 95 % CI , 63 - 80 ; P < 0 . 0001 ) and 114 min ( 95 % CI , 99 - 129 ; P < 0 . 0001 ; Welch ' s t - test ) , respectively .

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Figure 4(B) Analysis of cells containing wild-type Spo13, Spo13-mD, or Spo13-mD-m2. Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. spo13-mD delays Spc72 removal and Mpc70 loading by 30 min (95% CI, 26-34; P < 0.0001) and 60 min (95% CI, 55-66; P < 0.0001; Welch's t-test), respectively. (C) Analysis of cells expressing Clb1-mDK and different versions of Spo13. Meiotic events were quantified as in (B). spo13-mD plus PEST-clb1-mDK delays Spc72 removal and Mpc70 loading by 72 min (95% CI, 63-80; P < 0.0001) and 114 min (95% CI, 99-129; P < 0.0001; Welch's t-test), respectively.


Figure 5C . Cdc5 interacts with Ime2 . Cdc5 - ha3 , Ime2 , and Spo13 were detected by immunoblotting in whole - cell extracts and α - Ha immunoprecipitates from the indicated strains . Data information : data are representative of two independent experiments . D . Cdc5 ' s polo - box domain ( PBD ) binds to Ime2 . Ha3 - tagged versions of the wild - type PBD or the phosphopeptide - binding mutant PBD - FAA were expressed at t = 5 . 5 h in SPM in cells depleted of Cdc20 and Cdc5 . PBD - ha3 and Ime2 were detected by immunoblotting in whole - cell extracts and α - Ha immunoprecipitates . Data information : data are representative of two independent experiments . E . Cdc5 - dependent modification of Ime2 . PHSL1 - CDC20 ama1 ∆ control cells and cells containing spo13 ∆ or cdc5 - as were treated with CMK ( to inhibit Cdc5 - as ) at t = 8 h in SPM ( arrow heads ) . The panel shows immunoblot analysis of whole - cell extracts . C , sample from proliferating cells . Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5 - as . The arrow marks the modified form of Ime2 . Data information : data are representative of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C. Cdc5 interacts with Ime2. Cdc5-ha3, Ime2, and Spo13 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates from the indicated strains. Data information: data are representative of two independent experiments.D. Cdc5's polo-box domain (PBD) binds to Ime2. Ha3-tagged versions of the wild-type PBD or the phosphopeptide-binding mutant PBD-FAA were expressed at t = 5.5 h in SPM in cells depleted of Cdc20 and Cdc5. PBD-ha3 and Ime2 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates. Data information: data are representative of two independent experiments.E. Cdc5-dependent modification of Ime2. PHSL1-CDC20 ama1∆ control cells and cells containing spo13∆ or cdc5-as were treated with CMK (to inhibit Cdc5-as) at t = 8 h in SPM (arrow heads). The panel shows immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5-as. The arrow marks the modified form of Ime2. Data information: data are representative of two independent experiments.


Figure 6C , D . Immunoblot analysis of whole - cell extracts . C , sample from proliferating cells . ( C ) Protein levels in the cultures shown in ( A ) . ( D ) Protein levels in the cultures shown in ( B ) .

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Figure 6C, D. Immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. (C) Protein levels in the cultures shown in (A). (D) Protein levels in the cultures shown in (B).


Figure 7A , B . Mitotic cdc20 - 3 cells were shifted to 36 ° C for 80 min . At t = 0 , cells were treated with estradiol to induce expression from the PEST promoter . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP . Frame width , 19 μm . Bottom , quantification of cells with Spc72 - GFP at SPBs . ( A ) Expression of Ime2 - ∆ C induces removal of Spc72 from SPBs , which is inhibited by co - expression of Spo13 . ( B ) Expression of Cdc5 plus Ime2 - ∆ C causes rapid removal of Spc72 , which is inhibited by co - expression of Spo13 . Data information : data are representative of three independent experiments . C . Mitotic cdc20 - 3 cells containing PEST - CDC5 plus PEST - IME2 - ∆ C and / or PEST - NDT80 were shifted to 36 ° C for 30 min and subsequently treated with estradiol ( t = 0 ) . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP . Frame width , 19 μm . Bottom , quantification of cells with Mpc70 - GFP at SPBs . Data information : data are representative of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, B. Mitotic cdc20-3 cells were shifted to 36°C for 80 min. At t = 0, cells were treated with estradiol to induce expression from the PEST promoter. Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Spc72-GFP. Frame width, 19 μm. Bottom, quantification of cells with Spc72-GFP at SPBs. (A) Expression of Ime2-∆C induces removal of Spc72 from SPBs, which is inhibited by co-expression of Spo13. (B) Expression of Cdc5 plus Ime2-∆C causes rapid removal of Spc72, which is inhibited by co-expression of Spo13. Data information: data are representative of three independent experiments.C. Mitotic cdc20-3 cells containing PEST-CDC5 plus PEST-IME2-∆C and/or PEST-NDT80 were shifted to 36°C for 30 min and subsequently treated with estradiol (t = 0). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Frame width, 19 μm. Bottom, quantification of cells with Mpc70-GFP at SPBs. Data information: data are representative of two independent experiments.


Figure 8C . PHSL1 - CDC20 ama1 ∆ cdc28 - as2 control cells and cells containing spo13 ∆ or cdc5 - as were treated with 1Na - PP1 at t = 3 h in SPM to inhibit Cdc28 - as2 . At t = 9 h in SPM , Cdc28 - as2 was activated by washing cells with cSPM lacking 1Na - PP1 ( washout ) . cdc5 - as cells were washed with cSPM plus CMK to activate Cdc28 - as2 and inhibit Cdc5 - as . Top , time - lapse series from the imaging of Mpc70 - GFP and RFP - tubulin . The weak , nuclear signal originates from Ndt80 - GFP . Bottom , quantification of spindle formation and Mpc70 loading to SPBs . D . PHSL1 - CDC20 ama1 ∆ cells containing SPC72 , spc72 - 7 , or spc72 - 7 plus cdc5 - as were shifted from 24 to 36 ° C at t = 4 . 2 h in SPM ( to inactivate Spc72 - 7 , arrow heads ) and treated with CMK at t = 6 h in SPM ( to inhibit Cdc5 - as ) . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP . Bottom , quantification of SPB separation and Mpc70 loading to SPBs . Inhibition of Cdc5 - as in spc72 - 7 cells advances the time of Mpc70 loading by 119 min ( 95 % CI , 76 - 162 ; P < 0 . 0001 ; Welch ' s t - test ) . Data are representative of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8C. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing spo13∆ or cdc5-as were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 9 h in SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus CMK to activate Cdc28-as2 and inhibit Cdc5-as. Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs. D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5-as in spc72-7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.


Figure 9A . Inhibition of Cdk1 at anaphase I . CDC20 - mAR and CDC20 - mAR cdc28 - as1 cells were released from the metaphase I - arrest with CuSO4 at 7 h in SPM ( t = 0 ) and treated with 1NM - PP1 at t = 40 min to inhibit Cdc28 - as1 ( arrows ) . Top , time - lapse series from the imaging of Mpc70 - GFP , SPBs ( Cnm67 - RFP ) , and nuclei ( TetR - RFP ) . Bottom , quantification of meiotic events .

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Figure 9A. Inhibition of Cdk1 at anaphase I. CDC20-mAR and CDC20-mAR cdc28-as1 cells were released from the metaphase I-arrest with CuSO4 at 7 h in SPM (t = 0) and treated with 1NM-PP1 at t = 40 min to inhibit Cdc28-as1 (arrows). Top, time-lapse series from the imaging of Mpc70-GFP, SPBs (Cnm67-RFP), and nuclei (TetR-RFP). Bottom, quantification of meiotic events.


Figure 1Multiple tissue ( eye , brain , adrenal gland , submandibular gland , oesophagus , lung , heart , liver , kidney , spleen , pancreas , stomach , duodenum , jejunum , ileum , caecum , colon , spinal cord , dorsal root ganglia , skeletal muscle , testis , epididymis , female gonad , skull ) northern blot for CAVII . In the P56 mouse , CAVII is prominently expressed in the central nervous system including brain and spinal cord . Gapdh served as the loading control . ( C ) Developmental profile of CAVII protein expression . Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30 . The band of the appropriate size ( ∼ 30 kDa ) was absent in protein lysates from Car7 KO tissue . beta Actin served as the loading control in C and D . ( D ) In western blots from protein lysates of cultured cells , CAVII was detected in mixed glial / neuron cultures but not in glia cell cultures . The presence of astrocytes in all cultures was confirmed by detection of GFAP . Actin served as the loading control in C and D .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Multiple tissue (eye, brain, adrenal gland, submandibular gland, oesophagus, lung, heart, liver, kidney, spleen, pancreas, stomach, duodenum, jejunum, ileum, caecum, colon, spinal cord, dorsal root ganglia, skeletal muscle, testis, epididymis, female gonad, skull) northern blot for CAVII. In the P56 mouse, CAVII is prominently expressed in the central nervous system including brain and spinal cord. Gapdh served as the loading control.(C) Developmental profile of CAVII protein expression. Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30. The band of the appropriate size (∼30 kDa) was absent in protein lysates from Car7 KO tissue.beta Actin served as the loading control in C and D.(D) In western blots from protein lysates of cultured cells, CAVII was detected in mixed glial/neuron cultures but not in glia cell cultures. The presence of astrocytes in all cultures was confirmed by detection of GFAP. Actin served as the loading control in C and D.


Figure 2Original single - cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons ( baseline pHi 7 . 21 , 7 . 13 and 7 . 11 , respectively ) . Superfusion with CO2 / HCO3 − - free HEPES solution ( upper horizontal bars ) evoked an intracellular alkalinization , which in P14 WT was large and suppressed by 100 μM acetazolamide ( AZ , lower horizontal bar ) , indicating the presence of CA activity ( see Supplementary Materials and methods ) . The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide ( a poorly permeant CA inhibitor ) . Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT ( scale bar 10 μm ) . Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity . Data from CA II KO and CA II / VII KO neurons were obtained only at > P35 . The number of cells tested is indicated for each bar . The animal numbers for WT mice at the different age points was n = 3 ( P5 - 8 ) , n = 2 ( P10 - 11 ) , n = 3 ( P14 - 16 ) , n = 2 ( P17 - 18 ) , n = 2 ( P20 - 30 ) and n = 2 ( > P35 ) ; for CAVII KO mice the numbers were n = 3 ( P5 - 8 ) , n = 2 ( P10 - 11 ) , n = 5 ( P14 - 16 ) , n = 3 ( P17 - 18 ) , n = 4 ( P20 - 30 ) and n = 2 ( > P35 ) ; and for CA II KO and CA II / CAVII KO mice the number was n = 2 ( > P35 ) . Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2 / HCO3 − ( horizontal bar , HEPES ) in slices from > P35 WT , CAVII KO and CA II / VII KO mice . The rate of rise and the amplitude of the alkaline shift are identical in WT ( P39 , baseline pHi 7 . 01 ) and CAVII KO neurons ( P40 , 7 . 06 ) , whereas alkalinization develops much more slowly in the CA II / VII KO ( P46 , 7 . 04 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Original single-cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons (baseline pHi 7.21, 7.13 and 7.11, respectively). Superfusion with CO2/HCO3−-free HEPES solution (upper horizontal bars) evoked an intracellular alkalinization, which in P14 WT was large and suppressed by 100 μM acetazolamide (AZ, lower horizontal bar), indicating the presence of CA activity (see Supplementary Materials and methods). The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide (a poorly permeant CA inhibitor). Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT (scale bar 10 μm).Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity. Data from CA II KO and CA II/VII KO neurons were obtained only at >P35. The number of cells tested is indicated for each bar. The animal numbers for WT mice at the different age points was n=3 (P5-8), n=2 (P10-11), n=3 (P14-16), n=2 (P17-18), n=2 (P20-30) and n=2 (>P35); for CAVII KO mice the numbers were n=3 (P5-8), n=2 (P10-11), n=5 (P14-16), n=3 (P17-18), n=4 (P20-30) and n=2 (>P35); and for CA II KO and CA II/CAVII KO mice the number was n=2 (>P35).Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2/HCO3− (horizontal bar, HEPES) in slices from >P35 WT, CAVII KO and CA II/VII KO mice. The rate of rise and the amplitude of the alkaline shift are identical in WT (P39, baseline pHi 7.01) and CAVII KO neurons (P40, 7.06), whereas alkalinization develops much more slowly in the CA II/VII KO (P46, 7.04)


Figure 3Excitatory GABAergic response in WT , but not in CAVII KO neurons . Picrotoxin blocked the biphasic response ( PiTX 90 μM ; n = 3 / genotype ) . The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO ( 13 . 53 ± 1 . 27 versus 6 . 39 ± 0 . 96 mV / s , P < 0 . 001 , Student ' s t - test ) . The resting membrane potential of WT and CAVII KO neurons did not differ ( − 71 . 0 ± 1 . 1 versus − 72 . 3 ± 1 . 4 mV , respectively ; n = 15 neurons for both genotypes , P = 0 . 47 , Student ' s t - test ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D ) Microinjection of GABA ( 5 mM for 100 ms , sr / slm border in the presence of CGP55845 / TTX , arrowheads ) induced a pronounced depolarization in WT neurons ( black ) that was abolished in the absence of CO2 / HCO3 − ( grey ) . The depolarization was smaller in CA VII KO ( red ) . Bar diagram illustrates GABAergic mean peak depolarization in P12 - 16 WT and CAVII KO ( 13 . 3 ± 0 . 5 versus 8 . 3 ± 0 . 6 mV , P < 0 . 001 , Student ' s t - test ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D ) . In the presence of AP5 / CNQX / CGP55845 , microinjection of GABA ( 5 mM , sr / slm border ) triggered SR 95531 - sensitive field potential spikes in WT slices already upon 8 - ms injection , while even 16 - ms injection evoked hardly any spikes in CAVII KO ( left ) . Mean number of spikes was plotted against GABA puff duration in P12 - 16 WT and CAVII KO ( P < 0 . 01 and P < 0 . 001 , Student ' s t - test ; right ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D ) After P35 , depolarizations upon GABA microinjection ( arrowheads ) in the presence of CGP55845 / TTX were indistinguishable in WT ( black ) , CA VII KO ( red ) and CA II KO ( blue ) but smaller in CA II / VII KO ( green ) . The depolarization in WT was abolished in the absence of CO2 / HCO3 − ( grey , n = 5 ) . Bar diagram summarizes mean peak depolarization for each genotype ( right ; ANOVA , Bonferroni ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Excitatory GABAergic response in WT, but not in CAVII KO neurons. Picrotoxin blocked the biphasic response (PiTX 90 μM; n=3/genotype). The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO (13.53±1.27 versus 6.39±0.96 mV/s, P<0.001, Student's t-test). The resting membrane potential of WT and CAVII KO neurons did not differ (−71.0±1.1 versus −72.3±1.4 mV, respectively; n=15 neurons for both genotypes, P=0.47, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)Microinjection of GABA (5 mM for 100 ms, sr/slm border in the presence of CGP55845/TTX, arrowheads) induced a pronounced depolarization in WT neurons (black) that was abolished in the absence of CO2/HCO3− (grey). The depolarization was smaller in CA VII KO (red). Bar diagram illustrates GABAergic mean peak depolarization in P12-16 WT and CAVII KO (13.3±0.5 versus 8.3±0.6 mV, P<0.001, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D).In the presence of AP5/CNQX/CGP55845, microinjection of GABA (5 mM, sr/slm border) triggered SR 95531-sensitive field potential spikes in WT slices already upon 8-ms injection, while even 16-ms injection evoked hardly any spikes in CAVII KO (left). Mean number of spikes was plotted against GABA puff duration in P12-16 WT and CAVII KO (P<0.01 and P<0.001, Student's t-test; right). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)After P35, depolarizations upon GABA microinjection (arrowheads) in the presence of CGP55845/TTX were indistinguishable in WT (black), CA VII KO (red) and CA II KO (blue) but smaller in CA II/VII KO (green). The depolarization in WT was abolished in the absence of CO2/HCO3− (grey, n=5). Bar diagram summarizes mean peak depolarization for each genotype (right; ANOVA, Bonferroni). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)


Figure 4Representative raw traces of epidural EEG recordings during hyperthermia ( HT ) from a WT and a CA VII KO mouse . Seizures were reliably induced in WT ( n = 9 ) but not in CA VII KO ( n = 7 ) . Error bars denote s . e . m . , all P - values are based on Student ' s t - test ( * * * P < 0 . 001 ) , and animal number is given in bar diagrams . Rectal temperatures are given above the traces . Specimen recordings of breath rate from a WT and a CA VII KO mouse . The baseline breath rate ( WT 153 ± 11 breaths / min and CA VII KO 157 ± 8 breaths / min ) was increased during HT in both genotypes ( WT 272 ± 28 breaths / min and CA VII KO 279 ± 12 breaths / min ) . The bar diagram summarizes the mean increase in breath rate in WT ( 177 ± 12 % ) and CA VII KO mice ( 178 ± 6 % , P = 0 . 98 ) . Error bars denote s . e . m . , all P - values are based on Student ' s t - test ( * * * P < 0 . 001 ) , and animal number is given in bar diagrams . Rectal temperatures are given above the traces . Hyperthermia caused a comparable alkalosis in blood pH in both WT and CA VII KO mice . The blood pH did not differ between the two genotypes under control conditions ( P = 0 . 37 ) or during HT ( P = 0 . 44 ) . Error bars denote s . e . m . , all P - values are based on Student ' s t - test ( * * * P < 0 . 001 ) , and animal number is given in bar diagrams . Rectal temperatures are given above the traces . Electrographic seizures were not detected in WT mice when hyperthermia was induced in the continuous presence of 5 % CO2 ( n = 5 ) . Error bars denote s . e . m . , all P - values are based on Student ' s t - test ( * * * P < 0 . 001 ) , and animal number is given in bar diagrams . Rectal temperatures are given above the traces .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative raw traces of epidural EEG recordings during hyperthermia (HT) from a WT and a CA VII KO mouse. Seizures were reliably induced in WT (n=9) but not in CA VII KO (n=7). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.Specimen recordings of breath rate from a WT and a CA VII KO mouse. The baseline breath rate (WT 153±11 breaths/min and CA VII KO 157±8 breaths/min) was increased during HT in both genotypes (WT 272±28 breaths/min and CA VII KO 279±12 breaths/min). The bar diagram summarizes the mean increase in breath rate in WT (177±12%) and CA VII KO mice (178±6%, P=0.98).Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.Hyperthermia caused a comparable alkalosis in blood pH in both WT and CA VII KO mice. The blood pH did not differ between the two genotypes under control conditions (P=0.37) or during HT (P=0.44). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.Electrographic seizures were not detected in WT mice when hyperthermia was induced in the continuous presence of 5% CO2 (n=5). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Figure 5Diazepam ( DZP , 150 μg / kg ) reduced the latency to seizure onset in P14 rat pups ( P < 0 . 05 ) . There was no statistical difference between 50 μg / kg and saline - injected animals . DZP 2 . 5 mg / kg completely blocked the seizures . DZP was given intraperitoneally 15 min before hyperthermia ( HT ) onset . Diazepam given at 150 μg / kg did not affect the baseline breath rate , and HT induced an analogous increase in the breath rate in saline - and diazepam - injected rats ( P > 0 . 05 ) . At a higher concentration ( 2 . 5 mg / kg ) , DZP suppressed breathing significantly ( P < 0 . 01 ) and HT caused only a small increase in the breath rate . In slice preparation , DZP ( 1 μM ) potentiated the high - frequency stimulation ( 40 pulses at 100 Hz , horizontal bar ) - evoked GABAAR - mediated depolarization . Sample trace from a slice from a P14 rat pup where stimulation to the border of stratum radiatum and stratum lacunosum - moleculare was given in the presence of AP5 , CNQX and CGP 55845 . The bar diagrams summarize the effects of DZP on the duration of the depolarization ( time to half - maximum ) and the number of action potentials associated with the depolarizing phase . P - values ( * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ) are based on ANOVA , Bonferroni ( A , B ) or Student ' s t - test ( C ) , and error bars denote s . e . m . The number of animals ( A , B ) or cells ( C ) is indicated in bar diagrams .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Diazepam (DZP, 150 μg/kg) reduced the latency to seizure onset in P14 rat pups (P<0.05). There was no statistical difference between 50 μg/kg and saline-injected animals. DZP 2.5 mg/kg completely blocked the seizures. DZP was given intraperitoneally 15 min before hyperthermia (HT) onset.Diazepam given at 150 μg/kg did not affect the baseline breath rate, and HT induced an analogous increase in the breath rate in saline- and diazepam-injected rats (P>0.05). At a higher concentration (2.5 mg/kg), DZP suppressed breathing significantly (P<0.01) and HT caused only a small increase in the breath rate.In slice preparation, DZP (1 μM) potentiated the high-frequency stimulation (40 pulses at 100 Hz, horizontal bar) -evoked GABAAR-mediated depolarization. Sample trace from a slice from a P14 rat pup where stimulation to the border of stratum radiatum and stratum lacunosum-moleculare was given in the presence of AP5, CNQX and CGP 55845. The bar diagrams summarize the effects of DZP on the duration of the depolarization (time to half-maximum) and the number of action potentials associated with the depolarizing phase. P-values (*P<0.05, **P<0.01 and ***P<0.001) are based on ANOVA, Bonferroni (A, B) or Student's t-test (C), and error bars denote s.e.m. The number of animals (A, B) or cells (C) is indicated in bar diagrams.


Figure 6CA VII expression during human brain development . Line plots show the log2 - transformed exon array signal intensity in the neocortex ( NCX ) and hippocampal formation ( HIP ) from the early fetal period to late adulthood . The solid line between periods 7 and 8 separates the prenatal from the postnatal period . CA VII expression during human brain development . Line plots show the log . Line plots show the normalized ratio of absolute copy number of CA VII to Gapdh by droplet digital PCR ( ddPCR , right ) in the neocortex ( NCX ) and hippocampus ( HIP ) from the early fetal period to late adulthood . The solid line between periods 7 and 8 separates the prenatal from the postnatal period .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6CA VII expression during human brain development. Line plots show the log2-transformed exon array signal intensity in the neocortex (NCX) and hippocampal formation (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.CA VII expression during human brain development. Line plots show the log. Line plots show the normalized ratio of absolute copy number of CA VII to Gapdh by droplet digital PCR (ddPCR, right) in the neocortex (NCX) and hippocampus (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.


Figure 1 ( B ) Representative images of importin β cargo gradients detected by Rango - 3 FLIM / FRET imaging . Scale bars , 20µm . ( C ) Line scans of Rango - 3 FRET efficiency ( E ) in 10 µm - wide areas ( D - F ) Quantification of the Rango - 3 E gradients ( top panels ) and average cellular Rango - 3 E ( bottom ) in involving untreated oocytes ( controls ) or oocytes injected with RanT24N mRNA , importin β ( 71 - 876 ) protein or treated with 20 µM IPZ . Data for each panel are from at least 2 separate experiments . Means ± SDs , t - test , oocyte numbers indicated in brackets . ( G ) Nonlinear single - exponential regression was used to fit the average of radial Rango - 3 τdonor line scans in control oocyte with the one - phase decay model . The dashed line corresponds to the average of spindle pole distance from chromosomes in the same cell . ( H ) Immunoblotting in biochemical pull - downs with recombinant Ran proteins added to human DL D1 cell lysates . A representative of at least 5 repeats .

O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Representative images of importin β cargo gradients detected by Rango-3 FLIM/FRET imaging. Scale bars, 20µm.(C) Line scans of Rango-3 FRET efficiency (E) in 10 µm-wide areas(D-F) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) in involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20 µM IPZ. Data for each panel are from at least 2 separate experiments. Means ± SDs, t-test, oocyte numbers indicated in brackets.(G) Nonlinear single-exponential regression was used to fit the average of radial Rango-3 τdonor line scans in control oocyte with the one-phase decay model. The dashed line corresponds to the average of spindle pole distance from chromosomes in the same cell.(H) Immunoblotting in biochemical pull-downs with recombinant Ran proteins added to human DL D1 cell lysates. A representative of at least 5 repeats.


Figure 2 ( A ) Quantification of chromosomal cargo gradients ( top ) and average Rango - 3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations . Data from oocytes imaged side - by - side within one experiment . Means ± SDs , one - side ANOVA , Tukey ' s test . Numbers of analyzed oocytes are indicated in the brackets in the top panel and are identical for both panels . ( B ) Representative images of importin β cargo gradients detected by Rango - 3 FLIM / FRET imaging . Scale bars , 20µm . ( C ) Linescans of the estimated Rango - 3 FRET efficiency ( E ) in 10 µm - wide areas ( D ) Quantification of the Rango - 3 E gradients ( top panels ) and average cellular Rango - 3 E ( bottom ) oocytes that were treated side - by - side by the injection of mRNA for Rango - 3 , alongside with buffer ( controls ) or 2000ng / µl mRNA for RanT24N or RanT24N , T42A . Means ± SDs , one - side ANOVA , Tukey ' s test , oocyte numbers indicated in brackets .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Quantification of chromosomal cargo gradients (top) and average Rango-3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations. Data from oocytes imaged side-by-side within one experiment. Means ± SDs, one-side ANOVA, Tukey's test. Numbers of analyzed oocytes are indicated in the brackets in the top panel and are identical for both panels.(B) Representative images of importin β cargo gradients detected by Rango-3 FLIM/FRET imaging. Scale bars, 20µm.(C) Linescans of the estimated Rango-3 FRET efficiency (E) in 10 µm-wide areas(D) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) oocytes that were treated side-by-side by the injection of mRNA for Rango-3, alongside with buffer (controls) or 2000ng/µl mRNA for RanT24N or RanT24N, T42A. Means ± SDs, one-side ANOVA, Tukey's test, oocyte numbers indicated in brackets.


Figure 3 ( B ) Chromosomal gradients detected by RBP3 FLIM / FRET imaging . Scale bars , 20µm . ( C ) Line scans of the estimated RBP - 3 in 10 µm - wide areas ( D - F ) Quantification of the RBP - 3 gradients ( top panels ) and average cellular RBP - 3 E ( bottom ) in separate experiments involving untreated oocytes ( controls ) or oocytes injected with RanT24N mRNA , importin β ( 71 - 876 ) protein or treated with 20µM IPZ . Means ± SDs , t - test , oocyte numbers indicated in brackets . ( G ) Nonlinear single - exponential regression was used to fit the average of radial RBP - 3 τdonor line scans in control oocyte with the one - phase decay model . ( H ) Images of oocytes expressing GFP - Ran and RCC1 - mCherry from injected mRNAs before FRAP assays . Scale bars , 20 µm . ( I ) RCC1 - mCherry half - time fluorescence recovery ( right , means ± SDs , t - test . ) and an immobile fraction ( left , means ± SDs , Mann - Whitney - test ) in control and IPZ - treated cells , oocyte numbers indicated in brackets .

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Figure 3(B) Chromosomal gradients detected by RBP3 FLIM/FRET imaging. Scale bars, 20µm.(C) Line scans of the estimated RBP-3 in 10 µm-wide areas(D-F) Quantification of the RBP-3 gradients (top panels) and average cellular RBP-3 E (bottom) in separate experiments involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20µM IPZ. Means ± SDs, t-test, oocyte numbers indicated in brackets.(G) Nonlinear single-exponential regression was used to fit the average of radial RBP-3 τdonor line scans in control oocyte with the one-phase decay model.(H) Images of oocytes expressing GFP-Ran and RCC1-mCherry from injected mRNAs before FRAP assays. Scale bars, 20 µm.(I) RCC1-mCherry half-time fluorescence recovery (right, means ± SDs, t-test.) and an immobile fraction (left, means ± SDs, Mann-Whitney-test) in control and IPZ-treated cells, oocyte numbers indicated in brackets.