Figure 2 ( A ) Number of EPODs called in different NAP deletions . D . S . denotes genotypes where cells were collected in the deep stationary phase of growth . ( D ) Distributions of mean protein occupancy at WT EPOD boundaries and background . The blue dots denote the median and the black line displays the interquartile ranges in each condition . The dashed pink line represents the WT median . ( * ) indicate the Wilcoxon Rank Sum test p value comparing the change in pseudomedian vs . WT for each condition that has been adjusted using the Benjamini and Hochberg method ( against a null hypothesis of no difference in pseudomedians ) . The grey line denotes the same comparison between the D . S . conditions . Underlined ( * ) ' s indicate a gain in the pseudomedian compared to baseline conditions , while no underline indicates a loss in the pseudomedian compared to baseline conditions . p value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * * . Three biological replicates were used for the " WT , Rich " case and " WT ( deep stat . ) " case , and two biological replicates for the " WT , Min " and " WT ( 2 ) , Rich " conditions . ( E ) Protein occupancy over the waa operon . The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . The EPOD over the waa operon is lost in the ∆ stpA ∆ hns condition which results in increased accessibility and RNA polymerase occupancy ( G ) RNA - seq analysis shows log fold change compared to WT of waa operon expression upon deletion of hns and stpA . ( * ) indicate q value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * *

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 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 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 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 2(A) Number of EPODs called in different NAP deletions. D.S. denotes genotypes where cells were collected in the deep stationary phase of growth.(D) Distributions of mean protein occupancy at WT EPOD boundaries and background. The blue dots denote the median and the black line displays the interquartile ranges in each condition. The dashed pink line represents the WT median. (*) indicate the Wilcoxon Rank Sum test p value comparing the change in pseudomedian vs. WT for each condition that has been adjusted using the Benjamini and Hochberg method (against a null hypothesis of no difference in pseudomedians). The grey line denotes the same comparison between the D.S. conditions. Underlined (*)'s indicate a gain in the pseudomedian compared to baseline conditions, while no underline indicates a loss in the pseudomedian compared to baseline conditions. p value < 0.05 = *, <0.005=**, <0.0005=***. Three biological replicates were used for the "WT, Rich" case and "WT (deep stat.)" case, and two biological replicates for the "WT, Min" and "WT (2), Rich" conditions.(E) Protein occupancy over the waa operon. The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. The EPOD over the waa operon is lost in the ∆stpA∆hns condition which results in increased accessibility and RNA polymerase occupancy(G) RNA-seq analysis shows log fold change compared to WT of waa operon expression upon deletion of hns and stpA. (*) indicate q value < 0.05 = *, < 0.005 = **, < 0.0005 = ***


Figure 3 ( B ) Protein occupancy over the idn operon for each condition ( colors are denoted in ( A ) ) . The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . There is a large loss in protein occupancy when cells are shifted to 5KDG , leading to the loss of the call ed EPOD . Protein occupancy is restored once cells are grown in the second glucose condition . The 500bp normalized average of previously published H - NS ChIP - seq [ 20 ] exhibits high H - NS binding on the idnD promoter region . ( C ) To examine the expression of the idn operon at each shift , RNA - seq was performed . RNA - seq expression estimates log2 scaled for idn operon genes for WT cells grown in each condition ( as colored in ( A ) ) . Comparisons are denoted with colored dots with significance stars representing the adjusted p - value where ( * * * ) signify q - values < 0 .

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 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

Figure 3(B) Protein occupancy over the idn operon for each condition (colors are denoted in (A)). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. There is a large loss in protein occupancy when cells are shifted to 5KDG, leading to the loss of the call ed EPOD. Protein occupancy is restored once cells are grown in the second glucose condition. The 500bp normalized average of previously published H-NS ChIP-seq [20] exhibits high H-NS binding on the idnD promoter region.(C) To examine the expression of the idn operon at each shift, RNA-seq was performed. RNA-seq expression estimates log2 scaled for idn operon genes for WT cells grown in each condition (as colored in (A)). Comparisons are denoted with colored dots with significance stars representing the adjusted p-value where (***) signify q-values <0.0005


Figure 4 ( B ) Difference in Log2 ratio of fitnesses of the KDG - exposed cells with unexposed lacZ : : cat relative to that observed prior to KDG exposure , given as a function of time since exposure to KDG . Day - 1 corresponds to the day before KDG exposure , Day 0 is cells taken immediately after growth in KDG , and subsequent timepoints reflect different durations of growth in M9 Min Glu prior to competition ( See ( A ) ) . Points show medians across replicates ( 3 biological replicates for 0 . 5 days , 6 biological replicates for all other timepoints ) ; error bars show 95 % confidence intervals for the difference relative to the Day - 1 timepoint , calculated using the R wilcox . test function . Significance was assessed using a Wilcoxon rank sum test comparing the distribution at each timepoint to the - 1 day ( naive ) timepoint : * , p < 0 . 05 ; * * , p < 0 . 005 .

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

Figure 4(B) Difference in Log2 ratio of fitnesses of the KDG-exposed cells with unexposed lacZ::cat relative to that observed prior to KDG exposure, given as a function of time since exposure to KDG. Day -1 corresponds to the day before KDG exposure, Day 0 is cells taken immediately after growth in KDG, and subsequent timepoints reflect different durations of growth in M9 Min Glu prior to competition (See (A)). Points show medians across replicates (3 biological replicates for 0.5 days, 6 biological replicates for all other timepoints); error bars show 95% confidence intervals for the difference relative to the Day -1 timepoint, calculated using the R wilcox.test function. Significance was assessed using a Wilcoxon rank sum test comparing the distribution at each timepoint to the -1 day (naive) timepoint: *, p<0.05; **, p<0.005.


Figure 5 ( C ) Mean protein occupancy was calculated across all WT EPOD locations that contain annotated prophages . The change in mean protein occupancy compared to WT was calculated for each condition , where anything negative is a loss in occupancy compared to WT . Hierarchical clustering was performed to examine which genotypes clustered together and were more similar . Permutation tests comparing the change in occupancy over EPODs containing prophages were performed against the rest of the genome . P value < 0 . 05 = * , < 0 . 005 = * * indicate a negative mean change in occupancy compared to WT .

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

Figure 5(C) Mean protein occupancy was calculated across all WT EPOD locations that contain annotated prophages. The change in mean protein occupancy compared to WT was calculated for each condition, where anything negative is a loss in occupancy compared to WT. Hierarchical clustering was performed to examine which genotypes clustered together and were more similar. Permutation tests comparing the change in occupancy over EPODs containing prophages were performed against the rest of the genome. P value <0.05 = *, <0.005=** indicate a negative mean change in occupancy compared to WT.


Figure 6 ( A ) Example prophage region that is annotated with the Fis - and Hfq - associated HMM class 5 in our genome - wide HMM classification The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . Prophage genes are highlighted with a red box . The major peak associated with WT IPOD - HR occupancy is represented in a grey dashed line as a reference to compare IPOD - HR occupancy in the other genotypes . Modest loss of protein occupancy was observed at the same prophage - containing EPOD for ∆ fis ( light purple ; see color key in ( B ) ) and ∆ hfq ( dark purple ; see color key in ( B ) ) conditions compared to WT ( gold ) . ( B ) RNA - seq of WT , ∆ fis , and ∆ hfq were performed . The log fold change compared to WT was calculated at prophage genes contained in the dashed box in ( A ) . Induction of prophages across the region where loss in occupancy is observed . ( * ) indicate the adjusted p - value : < 0 . 10 = + , < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * * ( F ) Rate ratios of all genes ( grey ) and prophage genes ( red ) in each quadrant outline in ( E ) , showing a higher rate of genes that resided in the + / + category , indicating that many prophages are de - repressed in both ∆ fis and ∆ hfq . ( * ) indicate the p - value calculated from testing the null that the rate ratios are the same . P - value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * * ( G ) P1 vir transduction experiment to test the viability of ∆ fis and ∆ hfq . - Hfq indicates deleting Hfq and - CspE indicates deleting - CspE as a control . Strain identities are indicated in the box . Number of transductions were counted on LB + Kan plates ; all efficiencies are relative to WT , and thus the log - scaled relative transduction rate for the WT itself is 0 by definition . - R1 indicates that the prophage region in ( A ) was deleted to test whether the loss of prophages silenced by Fis and Hfq restored viability of a ∆ fis ∆ hfq genotype . R2 and R3 were other regions in the genome that contained prophages that appeared to have Fis / Hfq dependent EPODs . Plotted values are mean efficiencies across replicates , with error bars showing a 95 % credible interval obtained via Bayesian inference , assuming the replicate - level colony counts are Poisson distributed with a ( conjugate ) Gamma ( 0 , 0 ) prior ; all log ratios ( including error bounds ) are plotted relative to the mean WT value . Data obtained from 5 biological replicates for hfq : : kan transductions and 4 biological replicates for cspE : : kan transductions .

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 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 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 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

Figure 6(A) Example prophage region that is annotated with the Fis- and Hfq-associated HMM class 5 in our genome-wide HMM classification The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. Prophage genes are highlighted with a red box. The major peak associated with WT IPOD-HR occupancy is represented in a grey dashed line as a reference to compare IPOD-HR occupancy in the other genotypes. Modest loss of protein occupancy was observed at the same prophage-containing EPOD for ∆fis (light purple; see color key in (B)) and ∆hfq (dark purple; see color key in (B)) conditions compared to WT (gold).(B) RNA-seq of WT, ∆fis, and ∆hfq were performed. The log fold change compared to WT was calculated at prophage genes contained in the dashed box in (A). Induction of prophages across the region where loss in occupancy is observed. (*) indicate the adjusted p-value: <0.10 = +, < 0.05 = *, <0.005=**, <0.0005=***(F) Rate ratios of all genes (grey) and prophage genes (red) in each quadrant outline in (E), showing a higher rate of genes that resided in the +/+ category, indicating that many prophages are de-repressed in both ∆fis and ∆hfq. (*) indicate the p-value calculated from testing the null that the rate ratios are the same. P-value < 0.05 = *, <0.005=**, <0.0005=***(G) P1 vir transduction experiment to test the viability of ∆fis and ∆hfq. -Hfq indicates deleting Hfq and -CspE indicates deleting -CspE as a control. Strain identities are indicated in the box. Number of transductions were counted on LB + Kan plates; all efficiencies are relative to WT, and thus the log-scaled relative transduction rate for the WT itself is 0 by definition. -R1 indicates that the prophage region in (A) was deleted to test whether the loss of prophages silenced by Fis and Hfq restored viability of a ∆fis ∆hfq genotype. R2 and R3 were other regions in the genome that contained prophages that appeared to have Fis/Hfq dependent EPODs. Plotted values are mean efficiencies across replicates, with error bars showing a 95% credible interval obtained via Bayesian inference, assuming the replicate-level colony counts are Poisson distributed with a (conjugate) Gamma(0,0) prior; all log ratios (including error bounds) are plotted relative to the mean WT value. Data obtained from 5 biological replicates for hfq::kan transductions and 4 biological replicates for cspE::kan transductions.


Figure 7 ( A ) IPOD - HR ChIP - subtracted z - scores and RNA polymerase ChIP in the vicinity of an extended protein occupancy domain ( EPOD ) . ChIP tracks are shown as log2 extracted / input ratios ; the z - score is the ChIP - subtracted robust z - score smoothed with a 512 bp rolling median . ( B ) IPOD - HR and RNA polymerase ChIP tracks in the vicinity of a negative EPOD ( nEPOD ) . ( C ) Effects of deletion of rok in the vicinity of an nEPOD ; Rok ChIP data from [ 70 ] shows a strong overlap with the nEPOD boundary , whereas that occupancy region is lost in Δrok cells . ( E ) Comparison of IPOD occupancy and SMC ChIP occupancy in the vicinity of several typical EPODs .

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

Figure 7(A) IPOD-HR ChIP-subtracted z-scores and RNA polymerase ChIP in the vicinity of an extended protein occupancy domain (EPOD). ChIP tracks are shown as log2 extracted/input ratios; the z-score is the ChIP-subtracted robust z-score smoothed with a 512 bp rolling median. (B) IPOD-HR and RNA polymerase ChIP tracks in the vicinity of a negative EPOD (nEPOD).(C) Effects of deletion of rok in the vicinity of an nEPOD; Rok ChIP data from[70] shows a strong overlap with the nEPOD boundary, whereas that occupancy region is lost in Δrok cells.(E) Comparison of IPOD occupancy and SMC ChIP occupancy in the vicinity of several typical EPODs.


Figure 1 ( A ) Autophagic markers from mixed midbrain gba + / + , gba + / − , and gba − / − cultures were analyzed via western blotting using antibodies as indicated . ( B ) Autophagic flux was analyzed in gba + / + and gba − / − midbrain neurons . Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting . β - actin was used as a loading control . ( C ) Densitometry analyzes of autophagy of ( B ) expressed as a ratio of LC3II / LC3I . Error bars , ± SEM . * p < 0 . 05 . ( D ) Accumulation of p62 / SQSTM1 was analyzed via immunoblotting . β - actin was used as a loading control . ( E ) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO . Proteins were extracted and analyzed via western blotting and stained with anti - ubiquitin antibodies . ( F ) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated . β - actin was used as a loading control . ( G ) Proteasomal activity ( chymotrypsin activity ) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase . RFU , relative luciferase units . Data represent the mean ± SEM ( n = 3 , each experiment contained triplicates for each genotype ) . * p < 0 . 05 .

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 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 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 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 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

Figure 1(A) Autophagic markers from mixed midbrain gba+/+, gba+/−, and gba−/− cultures were analyzed via western blotting using antibodies as indicated.(B) Autophagic flux was analyzed in gba+/+ and gba−/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control.(C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. *p < 0.05.(D) Accumulation of p62/SQSTM1 was analyzed via immunoblotting. β-actin was used as a loading control.(E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies.(F) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated. β-actin was used as a loading control.(G) Proteasomal activity (chymotrypsin activity) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase. RFU, relative luciferase units. Data represent the mean ± SEM (n = 3, each experiment contained triplicates for each genotype). *p < 0.05.


Figure 2 ( A ) Levels of soluble and insoluble α - synuclein from midbrain of P1 gba + / + and gba − / − mice . TX - 100 soluble and insoluble ( SDS / Urea ) factions were isolated and analyzed via western blotting with α - synuclein antibodies . β - actin was used as a loading control . ( B ) Sagittal brain stem sections from gba + / + , gba + / − , and gba − / − mice were stained with α - synuclein antibodies . Scale bar , 1 , 000 μm .

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

Figure 2(A) Levels of soluble and insoluble α-synuclein from midbrain of P1 gba+/+ and gba−/− mice. TX-100 soluble and insoluble (SDS/Urea) factions were isolated and analyzed via western blotting with α-synuclein antibodies. β-actin was used as a loading control.(B) Sagittal brain stem sections from gba+/+, gba+/−, and gba−/− mice were stained with α-synuclein antibodies. Scale bar, 1,000 μm.


Figure 3 ( A ) Neurons and astrocytes from gba + / + , gba + / − , and gba − / − mice were stained with TMRM . The mean florescence intensity in mitochondria was analyzed via confocal microscopy ( n = 3 , > 32 cells analyzed / experiment ) . ( B ) Neurons from gba + / + and gba − / − bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy . After 1 min , 1 μM oligomycin was added followed by 1 μM FCCP ( n = 5 , three cells analyzed / experiment ) . ( C ) Neurons from gba + / + and gba − / − incubated with 5 mM methyl pyruvate 5 min prior to imaging . Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy . After 90 s , 1 μM oligomycin was added , followed by 1 μM FCCP ( n = 3 , three cells analyzed / experiment ) . ( D ) As in ( C ) , except cells were bathed in 10 mM methyl succinate , ( n = 3 , four cells / experiment ) . All data in this figure represent the mean ± SEM , * p < 0 . 05 . Neurons from gba + / + and gba − / − incubated with 5 mM methyl pyruvate 5 min prior to imaging . Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy . After 90 s , 1 μM oligomycin was added , followed by 1 μM FCCP ( n = 3 , three cells analyzed / experiment ) .

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 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 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

Figure 3(A) Neurons and astrocytes from gba+/+, gba+/−, and gba−/− mice were stained with TMRM. The mean florescence intensity in mitochondria was analyzed via confocal microscopy (n = 3, >32 cells analyzed/experiment).(B) Neurons from gba+/+ and gba−/− bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 1 min, 1 μM oligomycin was added followed by 1 μM FCCP (n = 5, three cells analyzed/experiment).(C) Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).(D) As in (C), except cells were bathed in 10 mM methyl succinate, (n = 3, four cells/experiment). All data in this figure represent the mean ± SEM, *p < 0.05. Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).


Figure 4 ( A ) Oxygen consumption rates of gba + / + , gba + / − , and gba − / − mixed midbrain cultures . Basal oxygen consumption was measured over 3 min . The maximal ( uncoupled ) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A ( n = 3 , three runs / experiment ) . ( B ) Complex I - NADH : Ubiquinone reductase activity from gba + / + , gba + / − , and gba − / − brains expressed as a ratio to citrate synthase ( n = 3 ) . ( C ) Complex II - III : Succinate dehydeogenase , cytochome c reductase activity from gba + / + , gba + / − , and gba − / − brains expressed as a ratio to citrate synthase ( n = 3 ) . ( D ) Complex IV activity from gba + / + , gba + / − , and gba − / − brains expressed as a ratio to citrate synthase ( n = 3 ) . ( E ) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy ( n = 3 , > 16 cells / experiment ) . ( F ) gba + / + and gba − / − cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies . All data in this figure represent the mean ± SEM , * p < 0 . 05 .

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 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 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 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 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

Figure 4(A) Oxygen consumption rates of gba+/+, gba+/−, and gba−/− mixed midbrain cultures. Basal oxygen consumption was measured over 3 min. The maximal (uncoupled) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A (n = 3, three runs/experiment).(B) Complex I-NADH: Ubiquinone reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).(C) Complex II-III: Succinate dehydeogenase, cytochome c reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).(D) Complex IV activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).(E) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy (n = 3, >16 cells/experiment).(F) gba+/+and gba−/− cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies. All data in this figure represent the mean ± SEM, *p < 0.05.


Figure 5 ( A ) Neurons and astrocytes were immunostained for cytochrome c and stained with Hoechst . Far left panels were additionally immunostained for GFAP and MAP2 to mark neurons and astrocytes . " A " in nucleus denotes astrocytes . Scale bar , 20 μm . ( B ) Mitochondrial morphology was blind counted from cells in ( A ) with neurons scored as MAP2 positive and astrocytes as GFAP positive . A " normal " morphology is classified as a mixture of fused and fragmented mitochondria that comprise a wild - type network . ( n = 3 ; 100 cells counted / experiment ) . ( C ) Levels of morphology proteins from gba + / + , gba + / − , and gba − / − isolated mitochondria were analyzed via immunoblotting with the indicated antibodies . ( D ) Midbrain gba + / + , gba + / − , and gba − / − astrocytes expressing GFP - DRP1K38A were immunostained for cytochrome c and mitochondrial morphologies analyzed ; concurrently cells were stained with TMRM and fluorescence intensity analyzed via confocal microscopy . Scale bar , 20 μm ( n = 3 , > 15 cells / experiment ) . All data in this figure represent the mean ± SEM .

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 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 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

Figure 5(A) Neurons and astrocytes were immunostained for cytochrome c and stained with Hoechst. Far left panels were additionally immunostained for GFAP and MAP2 to mark neurons and astrocytes. "A" in nucleus denotes astrocytes. Scale bar, 20 μm.(B) Mitochondrial morphology was blind counted from cells in (A) with neurons scored as MAP2 positive and astrocytes as GFAP positive. A "normal" morphology is classified as a mixture of fused and fragmented mitochondria that comprise a wild-type network. (n = 3; 100 cells counted/experiment).(C) Levels of morphology proteins from gba+/+, gba+/−, and gba−/− isolated mitochondria were analyzed via immunoblotting with the indicated antibodies.(D) Midbrain gba+/+, gba+/−, and gba−/− astrocytes expressing GFP-DRP1K38A were immunostained for cytochrome c and mitochondrial morphologies analyzed; concurrently cells were stained with TMRM and fluorescence intensity analyzed via confocal microscopy. Scale bar, 20 μm (n = 3, >15 cells/experiment). All data in this figure represent the mean ± SEM.


Figure 6 ( A ) Midbrain astrocytes were transfected with GFP - LC3 , treated with 100 nM bafilomycin A1 , and stained with MitoTracker Red and imaged using confocal microscopy . ( B ) Midbrain gba + / + and gba − / − neurons and astrocytes were transfected with YFP - Parkin . Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy . Scale bar , 20 μm . ( C ) TMRM fluorescence intensity of cells from ( B ) was analyzed via confocal microscopy . Data represent the mean ± SEM , ( n = 3 , > 4 cells analyzed per experiment ) . ( D ) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr . PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba + / + and gba − / − neurons . Mitochondrial CI subunit NDUFS4 was used as a loading control .

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 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 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

Figure 6(A) Midbrain astrocytes were transfected with GFP-LC3, treated with 100 nM bafilomycin A1, and stained with MitoTracker Red and imaged using confocal microscopy.(B) Midbrain gba+/+ and gba−/− neurons and astrocytes were transfected with YFP-Parkin. Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy. Scale bar, 20 μm.(C) TMRM fluorescence intensity of cells from (B) was analyzed via confocal microscopy. Data represent the mean ± SEM, (n = 3, >4 cells analyzed per experiment).(D) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr. PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba+/+ and gba−/− neurons. Mitochondrial CI subunit NDUFS4 was used as a loading control.


Figure 1 ( A ) Representative immunofluorescence images of Polo - phospho ( ph ) - T182 levels at KTs of Drosophila neuroblasts throughout mitotic progression . Insets show magnifications of the outlined regions showing kinetochores ( KTs ) in early prometaphase ( EP ) , late prometaphase ( LP ) KTs and aligned KTs in metaphase ( M ) . Plotted profiles of signal intensities of phospho ( ph ) - T182 and the KT protein Spc105 are shown for the highlighted KTs . ( B ) Graph represents the levels of Polo phT182 at KTs for neuroblasts shown in ( A ) . phT182 signal was determined relative to Spc105 and all values normalized to the mean value determined for EP , which was set to 100 % ( n ≥ 67 KTs from at least 19 neuroblasts for each condition , n = 4 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( C ) Selected stills from live imaging analysis of mitotic progression in neuroblasts expressing UASPoloWT or UASPoloT182D under the control of inscuteable - Gal4 driver . Neuroblasts without Polo overexpression were used as control . Mitotic progression was followed in vivo by direct visualization of tubulin - RFP and the centromere marker CID - GFP . Time 0 refers to nuclear envelope breakdown ( NEBD ) . ( D ) Quantification of the mitotic time ( from NEBD to anaphase onset ) for neuroblasts shown in ( C ) . NEBD was identified as the time tubulin entered the nuclear space and anaphase onset as the time sister KTs separated ( n ≥ 12 neuroblasts for each condition , n ≥ 3 independent experiments ) ( E ) Quantification of the time spent in prometaphase ( from NEBD until last KT alignment at the metaphase plate ) for neuroblasts shown in ( C ) ( n ≥ 11 neuroblasts for each condition , n ≥ 3 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( F ) Measurement of inter - kinetochore distance during mitotic progression for neuroblasts shown in ( C ) . Only KT pairs within the maximum of two consecutive z - planes were considered eligible for quantification . Images were acquired every 20 sec . The graph shows the mean distance between two CID centroids in a KT pair over time ( t = 0 is NEBD ) . A linear regression was applied to the data set . Vertical dashed lines highlight the time at which cells overexpressing PoloWT ( gray ) or PoloT182D ( red ) reach the average inter - kinetochore distance measured in metaphase cells without Polo overexpression ( black ) ( n ≥ 7 neuroblasts for each condition ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( G ) Selected stills from live imaging of neuroblasts expressing Mad2 - GFP to follow KT - MT attachment status upon expression of PoloWT or PoloT182D . CID - RFP was used as KT reference . Insets show magnifications of the outlined regions showing single KT pairs that take longer to align at the metaphase plate . Asterisks indicate direction of chromosome segregation ( putative spindle pole positions ) . ( H ) Graph represents the mean fluorescence intensity ( MFI ) for Mad2 - GFP at KTs measured from NEBD to anaphase onset for neuroblasts shown in ( G ) . Each line represents the average of all KTs measured from a single neuroblast at each time point . Mad2 - GFP MFI was determined relative to CID - RFP MFI ( n ≥ 16 neuroblasts for each condition , n = 5 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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 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 O O O 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 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

Figure 1(A) Representative immunofluorescence images of Polo-phospho(ph)-T182 levels at KTs of Drosophila neuroblasts throughout mitotic progression. Insets show magnifications of the outlined regions showing kinetochores (KTs) in early prometaphase (EP), late prometaphase (LP) KTs and aligned KTs in metaphase (M). Plotted profiles of signal intensities of phospho(ph)-T182 and the KT protein Spc105 are shown for the highlighted KTs. (B) Graph represents the levels of Polo phT182 at KTs for neuroblasts shown in (A). phT182 signal was determined relative to Spc105 and all values normalized to the mean value determined for EP, which was set to 100% (n≥67 KTs from at least 19 neuroblasts for each condition, n=4 independent experiments).Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(C) Selected stills from live imaging analysis of mitotic progression in neuroblasts expressing UASPoloWT or UASPoloT182D under the control of inscuteable-Gal4 driver. Neuroblasts without Polo overexpression were used as control. Mitotic progression was followed in vivo by direct visualization of tubulin-RFP and the centromere marker CID-GFP. Time 0 refers to nuclear envelope breakdown (NEBD). (D) Quantification of the mitotic time (from NEBD to anaphase onset) for neuroblasts shown in (C). NEBD was identified as the time tubulin entered the nuclear space and anaphase onset as the time sister KTs separated (n≥12 neuroblasts for each condition, n≥3 independent experiments) (E) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (C) (n≥11 neuroblasts for each condition, n≥3 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(F) Measurement of inter-kinetochore distance during mitotic progression for neuroblasts shown in (C). Only KT pairs within the maximum of two consecutive z-planes were considered eligible for quantification. Images were acquired every 20 sec. The graph shows the mean distance between two CID centroids in a KT pair over time (t=0 is NEBD). A linear regression was applied to the data set. Vertical dashed lines highlight the time at which cells overexpressing PoloWT (gray) or PoloT182D (red) reach the average inter-kinetochore distance measured in metaphase cells without Polo overexpression (black) (n≥7 neuroblasts for each condition). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(G) Selected stills from live imaging of neuroblasts expressing Mad2-GFP to follow KT-MT attachment status upon expression of PoloWT or PoloT182D. CID-RFP was used as KT reference. Insets show magnifications of the outlined regions showing single KT pairs that take longer to align at the metaphase plate. Asterisks indicate direction of chromosome segregation (putative spindle pole positions). (H) Graph represents the mean fluorescence intensity (MFI) for Mad2-GFP at KTs measured from NEBD to anaphase onset for neuroblasts shown in (G). Each line represents the average of all KTs measured from a single neuroblast at each time point. Mad2-GFP MFI was determined relative to CID-RFP MFI (n≥16 neuroblasts for each condition, n=5 independent experiments).Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


Figure 2 ( A ) Representative immunofluorescence images of KT alignment efficiency in mitotic S2 cells expressing PoloWT - EGFP , PoloT182D - EGFP or lacking expression of any Polo transgene . Cells were treated with MG132 prior to fixation to prevent cells from exiting mitosis and increase the number of pre - anaphase figures . Insets display magnifications of the outlined regions , which highlight both low - tension misaligned ( inset 1 and 2 ) and high - tension aligned KTs ( inset 3 ) . ( B ) Graph represents the percentage of cells in each indicated mitotic state , as shown in ( A ) ( n ≥ 532 cells for each condition , n = 2 independent experiments ) . Data information : Data are shown as mean ± SD . Scale bar : 5 μm . ( C ) Representative immunofluorescence images of calcium - stable KT - MT attachments in metaphase S2 cells expressing either PoloWT - EGFP or PoloT182D - EGFP . Insets display magnifications of the outlined regions . Asterisk highlights either an aligned KT pair attached to MTs in an end - on fashion ( PoloWT - EGFP ) or an aligned KT pair in which a sister KT is laterally attached to the end of a MT fiber ( PoloT182D - EGFP ) . Plotted profiles show the overlap between Polo - EGFP and tubulin signals for the highlighted KT . ( D ) Graph represents the percentage of metaphase cells showing at least 1 KT with a lateral interaction , as shown in ( C ) ( asterisk ) ( n ≥ 58 cells for each condition , n = 2 independent experiments ) . Data information : Data are shown as mean ± SD . Scale bar : 5 μm . ( E ) Selected frames from live image analysis of chromosome segregation fidelity in neuroblasts expressing either PoloWT or PoloT182D . Neuroblasts without Polo overexpression were used as control . Jupiter - GFP was imaged for direct visualization of the mitotic spindle . Time 0 refers to anaphase onset . ( F ) Representative immunofluorescence images of mitotic neuroblasts from squashed brains showing distinct chromosome content . Spc105 was used as reference for KTs . Chromosome content is shown for each neuroblast . ( G ) Table shows the quantification of the percentage of asynchronous migration ( n ≥ 23 neuroblasts for each condition , n ≥ 3 independent experiments ) and of aneuploid cells respectively . For the quantification of aneuploidy , the whole brain was analyzed and prometaphase and metaphase neuroblasts were scored for chromosome number . An aneuploid cell was considered when > 8 chromosomes ( chr ) were visualized ( n ≥ 360 neuroblasts from at least 7 larvae brains for each condition , n ≥ 3 independent experiments ) . ND , not determined .

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 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 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 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 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

Figure 2(A) Representative immunofluorescence images of KT alignment efficiency in mitotic S2 cells expressing PoloWT-EGFP, PoloT182D-EGFP or lacking expression of any Polo transgene. Cells were treated with MG132 prior to fixation to prevent cells from exiting mitosis and increase the number of pre-anaphase figures. Insets display magnifications of the outlined regions, which highlight both low-tension misaligned (inset 1 and 2) and high-tension aligned KTs (inset 3). (B) Graph represents the percentage of cells in each indicated mitotic state, as shown in (A) (n≥ 532 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.(C) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Asterisk highlights either an aligned KT pair attached to MTs in an end-on fashion (PoloWT-EGFP) or an aligned KT pair in which a sister KT is laterally attached to the end of a MT fiber (PoloT182D-EGFP). Plotted profiles show the overlap between Polo-EGFP and tubulin signals for the highlighted KT. (D) Graph represents the percentage of metaphase cells showing at least 1 KT with a lateral interaction, as shown in (C) (asterisk) (n≥58 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.(E) Selected frames from live image analysis of chromosome segregation fidelity in neuroblasts expressing either PoloWT or PoloT182D. Neuroblasts without Polo overexpression were used as control. Jupiter-GFP was imaged for direct visualization of the mitotic spindle. Time 0 refers to anaphase onset.(F) Representative immunofluorescence images of mitotic neuroblasts from squashed brains showing distinct chromosome content. Spc105 was used as reference for KTs. Chromosome content is shown for each neuroblast.(G) Table shows the quantification of the percentage of asynchronous migration (n≥23 neuroblasts for each condition, n≥3 independent experiments) and of aneuploid cells respectively. For the quantification of aneuploidy, the whole brain was analyzed and prometaphase and metaphase neuroblasts were scored for chromosome number. An aneuploid cell was considered when >8 chromosomes (chr) were visualized (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). ND, not determined.


Figure 3 ( B ) Representative images of the eye phenotype observed in adult flies that were expressing UASPoloT182D in the eye imaginal disc during development at indicated temperatures . An example of an eye from an adult fly co - expressing UASPoloT182D with UASRodRNAi is also shown . Expression of UASlacZ transgene in the eye imaginal disc was used as control for overexpression and UAS dilution effect . ( C ) Graph represents the mean percentage of males born at 25 ° C either expressing eyGal4 - driven UASPoloT182D together with UASlacZ or in combination with UASRodRNAi ( n ≥ 3 independent crosses for each condition ) . The number of males with the genotype of interest born in each cross was normalized to the mean total number of males that are born in a control cross ( eyGal4 > UASlacZ , n = 9 independent crosses ) . Data information : Data are shown as mean ± SD . Scale bar : 5 μm . ( D ) Selected stills from live cell imaging of neuroblasts expressing either UASPoloT182D alone or in Rod - depleted background ( UASPoloT182D + UASRodRNAi ) . Neuroblasts without transgene expression were used as control and neuroblasts expressing UASPoloT182D together with UASlacZ were used as control for UAS dilution effect . ( E ) Quantification of the time spent in prometaphase ( from NEBD until last KT alignment at the metaphase plate ) for neuroblasts shown in ( D ) ( n ≥ 17 neuroblasts for each condition , n ≥ 4 independent experiments ) . Statistical analysis was calculated using an one - way ANOVA test for multiple comparisons . p values : ns , not significant ; * * , < 0 . 01 . Data information : Data are shown as mean ± SD . Scale bar : 5 μm . ( F ) Quantification of the percentage of prometaphase / metaphase mitotic figures per brain showing abnormal chromosome number ( > 8 chromosomes ( chr ) ( n ≥ 360 neuroblasts from at least 7 larvae brains for each condition , n ≥ 3 independent experiments ) . Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : ns , not significant ; * * , < 0 . 01 . Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

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 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 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 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

Figure 3(B) Representative images of the eye phenotype observed in adult flies that were expressing UASPoloT182D in the eye imaginal disc during development at indicated temperatures. An example of an eye from an adult fly co-expressing UASPoloT182D with UASRodRNAi is also shown. Expression of UASlacZ transgene in the eye imaginal disc was used as control for overexpression and UAS dilution effect.(C) Graph represents the mean percentage of males born at 25°C either expressing eyGal4-driven UASPoloT182D together with UASlacZ or in combination with UASRodRNAi (n≥3 independent crosses for each condition). The number of males with the genotype of interest born in each cross was normalized to the mean total number of males that are born in a control cross (eyGal4>UASlacZ, n=9 independent crosses). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.(D) Selected stills from live cell imaging of neuroblasts expressing either UASPoloT182D alone or in Rod-depleted background (UASPoloT182D+UASRodRNAi). Neuroblasts without transgene expression were used as control and neuroblasts expressing UASPoloT182D together with UASlacZ were used as control for UAS dilution effect. (E) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (D) (n≥17 neuroblasts for each condition, n≥4 independent experiments). Statistical analysis was calculated using an one-way ANOVA test for multiple comparisons. p values: ns, not significant; **, <0.01. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.(F) Quantification of the percentage of prometaphase/metaphase mitotic figures per brain showing abnormal chromosome number (>8 chromosomes (chr) (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; **, <0.01. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


Figure 4 ( A ) Selected stills from live cell imaging of neuroblasts expressing UASPoloWT , UASPoloT182D , or depleted of dynein heavy chain ( UASDhcRNAi ) and expressing Rod - GFP under control of its endogenous promoter . CID - RFP was used as a KT reference . Insets display magnifications of the outlined regions , which highlight late congressing KTs . Asterisks indicate direction of chromosome segregation ( putative spindle pole positions ) . ( B ) Graph represents the mean fluorescence intensity ( MFI ) for Rod - GFP determined relative to CID - RFP MFI for the KT pairs highlighted in ( A ) . Time was measured from the first frame a KT pair moved away from the other congressing KTs , until metaphase alignment ( vertical dashed lines ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( C ) Graph represents the levels of KT Rod - GFP determined relative to CID - RFP measured throughout mitotic progression for control neuroblasts ( UASPoloWT , n = 24 neuroblasts , n = 4 independent experiments ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( D ) Graph represents the levels of Rod - GFP determined relative to CID - RFP measured at late congressing KTs ( n ≥ 15 neuroblasts for each condition , n ≥ 3 independent experiments ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( E ) Selected stills from live imaging analysis of Rod - GFP streaming from KTs in neuroblasts expressing either PoloWT or PoloT182D . Asterisks indicate direction of chromosome segregation ( putative spindle poles positions ) . Arrowheads highlight streaming of Rod - GFP from unaligned KTs . Representative kymographs are shown for Rod - GFP streaming from metaphase until anaphase onset ( AO ) . Frames were acquired every 5sec . Data information : Scale bar : 5 μm .

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 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 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 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

Figure 4(A) Selected stills from live cell imaging of neuroblasts expressing UASPoloWT, UASPoloT182D, or depleted of dynein heavy chain (UASDhcRNAi) and expressing Rod-GFP under control of its endogenous promoter. CID-RFP was used as a KT reference. Insets display magnifications of the outlined regions, which highlight late congressing KTs. Asterisks indicate direction of chromosome segregation (putative spindle pole positions). (B) Graph represents the mean fluorescence intensity (MFI) for Rod-GFP determined relative to CID-RFP MFI for the KT pairs highlighted in (A). Time was measured from the first frame a KT pair moved away from the other congressing KTs, until metaphase alignment (vertical dashed lines). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(C) Graph represents the levels of KT Rod-GFP determined relative to CID-RFP measured throughout mitotic progression for control neuroblasts (UASPoloWT, n= 24 neuroblasts, n=4 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(D) Graph represents the levels of Rod-GFP determined relative to CID-RFP measured at late congressing KTs (n≥15 neuroblasts for each condition, n≥3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(E) Selected stills from live imaging analysis of Rod-GFP streaming from KTs in neuroblasts expressing either PoloWT or PoloT182D. Asterisks indicate direction of chromosome segregation (putative spindle poles positions). Arrowheads highlight streaming of Rod-GFP from unaligned KTs. Representative kymographs are shown for Rod-GFP streaming from metaphase until anaphase onset (AO). Frames were acquired every 5sec. Data information: Scale bar: 5 μm.


Figure 5 ( A ) Representative immunofluorescence images of ZW10 levels at unaligned KTs in neuroblasts expressing UASPoloWT , UASPoloT182D or depleted of dynactin subunit p50 ( UASp50RNAi ) or Spindly ( UASSpindlyRNAi ) . Spc105 was used as a KT reference . Insets display magnifications of the outlined regions which highlight both late congressing KT ( inset 1 ) and sister KTs that are non - oriented along the spindle axis ( inset 2 , except for the UASPoloWT panel which displays congressed KTs ) . UASPoloWT neuroblasts were used as control . Data information : Scale bar : 5 μm . ( B ) Graph represents ZW10 levels at late congressing and non - oriented KTs for neuroblasts Cells were selected when most KTs were congressed and late congressing KTs were clearly identified . Similarly , non - oriented KTs were considered when the KT - KT axis was not parallel to most congressed KTs ( or a metaphase plate ) . ZW10 signal was determined relative to Spc105 and all values were normalized to the control mean fluorescence intensity , which was set to 100 % ( n ≥ 69 KTs from at least 19 neuroblasts for each condition , n ≥ 3 independent experiments ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * , < 0 . 05 ; * * , < 0 . 01 ; * * * , < 0 . 001 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . ( C ) Selected stills from live imaging analysis of KT alignment in neuroblasts expressing UASPoloT182D , either alone or in a Rod - depleted background ( UASPoloT182D + UASRodRNAi ) , depleted of dynactin ( UASp50RNAi ) , Spindly ( UASSpindlyRNAi ) , dynein ( UASDhcRNAi ) or Rod in a dynein - depleted background ( UASDhcRNAi + UASRodRNAi ) . Outlined regions show KTs that are non - oriented ( lateral attachment ) relative to spindle axis and that were followed over time until stable biorientation ( arrowhead ) . Cartoon depicts the behavior of the respective KT pair over successive frames relative to the spindle axis ( dashed lines ) . Neuroblasts without transgene expression were used as control . Data information : Scale bar : 5 μm . ( D ) Quantification of the time a KT pair takes from the first contact with MTs until stable biorientation for neuroblasts ( n ≥ 12 KTs from at least 8 neuroblasts for each condition , n ≥ 5 independent experiments ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * , < 0 . 05 ; * * , < 0 . 01 ; * * * , < 0 . 001 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD .

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 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 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

Figure 5(A) Representative immunofluorescence images of ZW10 levels at unaligned KTs in neuroblasts expressing UASPoloWT, UASPoloT182D or depleted of dynactin subunit p50 (UASp50RNAi) or Spindly (UASSpindlyRNAi). Spc105 was used as a KT reference. Insets display magnifications of the outlined regions which highlight both late congressing KT (inset 1) and sister KTs that are non-oriented along the spindle axis (inset 2, except for the UASPoloWT panel which displays congressed KTs). UASPoloWT neuroblasts were used as control. Data information: Scale bar: 5 μm.(B) Graph represents ZW10 levels at late congressing and non-oriented KTs for neuroblasts Cells were selected when most KTs were congressed and late congressing KTs were clearly identified. Similarly, non-oriented KTs were considered when the KT-KT axis was not parallel to most congressed KTs (or a metaphase plate). ZW10 signal was determined relative to Spc105 and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥69 KTs from at least 19 neuroblasts for each condition, n≥3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: *, <0.05; **, <0.01; ***, <0.001; ****, <0.0001. Data are shown as mean ± SD.(C) Selected stills from live imaging analysis of KT alignment in neuroblasts expressing UASPoloT182D, either alone or in a Rod-depleted background (UASPoloT182D+UASRodRNAi), depleted of dynactin (UASp50RNAi), Spindly (UASSpindlyRNAi), dynein (UASDhcRNAi) or Rod in a dynein-depleted background (UASDhcRNAi+UASRodRNAi). Outlined regions show KTs that are non-oriented (lateral attachment) relative to spindle axis and that were followed over time until stable biorientation (arrowhead). Cartoon depicts the behavior of the respective KT pair over successive frames relative to the spindle axis (dashed lines). Neuroblasts without transgene expression were used as control. Data information: Scale bar: 5 μm.(D) Quantification of the time a KT pair takes from the first contact with MTs until stable biorientation for neuroblasts (n≥12 KTs from at least 8 neuroblasts for each condition, n≥5 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: *, <0.05; **, <0.01; ***, <0.001; ****, <0.0001. Data are shown as mean ± SD.


Figure 6 ( A ) Representative immunofluorescence images of Spindly localization to unaligned KTs in Drosophila S2 cells expressing either PoloWT - EGFP or PoloT182D - EGFP . Insets display magnifications of the outlined regions . Cells were treated with MG132 prior to fixation to increase the number of late prometaphase figures and allow better identification of late congressing KTs . CID was used as a KT reference . ( B ) Graph represents the percentage of Spindly levels at late congressing and non - oriented ( not oriented parallel to the spindle axis ) KTs for cells shown in ( A ) . Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in cells expressing PoloWT - EGFP , which was set to 100 % ( n ≥ 121 KTs from at least 39 cells for each condition , n = 3 independent experiments ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( C ) Selected frames from live imaging analysis of SpindlyWT - EGFP behavior during KT alignment in Drosophila S2 cells expressing either PoloWT - mRFP or PoloT182D - mRFP . Insets show magnifications of the outlined regions that highlight KTs with delayed congression to the spindle equator ( yellow dashed line ) . Time 0 is the first shift in KT direction from poleward to anti - poleward movement . ( D ) Graph represents the variation in SpindlyWT - EGFP levels at KTs highlighted in ( C ) and its correlation with efficiency of alignment . Spindly - EGFP mean fluorescence intensity ( MFI ) levels were corrected for photobleaching during acquisition and normalized to the first frame of analysis to account for differences in expression levels . KTs were tracked during the process of congression ( using Polo - mRFP signal as a KT reference ) and the distance from the unaligned KT pair to the spindle equator was measured . All values were normalized to the last frame of analysis ( stable integration in metaphase plate ) . Time 0 is the first shift in KT direction from poleward to anti - poleward movement ( n ≥ 9 KTs from at least 5 cells for each condition , n ≥ 4 independent experiments ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( E ) Representative immunofluorescence images of Spindly accumulation at aligned KTs in Drosophila S2 cells expressing either PoloWT - EGFP or PoloT182D - EGFP , in control or upon depletion of dynein heavy chain . Insets display magnifications of the outlined regions . CID was used as a KT reference . Data information : Scale bar : 5 μm . ( F ) Representative immunofluorescence images of Spindly accumulation at unattached KTs in Drosophila S2 cells expressing either PoloWT - EGFP or PoloT182D - EGFP . Insets display magnifications of the outlined regions . Cells were treated with colchicine prior to fixation to generate unattached KTs . CID was used as a KT reference . Data information : Scale bar : 5 μm . ( G ) Graph represents Spindly levels at aligned KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in control PoloWT - EGFP expressing cells , which was set to 100 % ( n ≥ 633 KTs from at least 35 cells for each condition , n = 2 independent experiments ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( H ) Graph represents Spindly levels at unattached KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in PoloWT - EGFP expressing cells , which was set to 100 % ( n ≥ 1282 KTs from at least 59 cells for each condition , n = 2 independent experiments ) . Data information : Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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

Figure 6(A) Representative immunofluorescence images of Spindly localization to unaligned KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Cells were treated with MG132 prior to fixation to increase the number of late prometaphase figures and allow better identification of late congressing KTs. CID was used as a KT reference. (B) Graph represents the percentage of Spindly levels at late congressing and non-oriented (not oriented parallel to the spindle axis) KTs for cells shown in (A). Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in cells expressing PoloWT-EGFP, which was set to 100% (n≥121 KTs from at least 39 cells for each condition, n=3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(C) Selected frames from live imaging analysis of SpindlyWT-EGFP behavior during KT alignment in Drosophila S2 cells expressing either PoloWT-mRFP or PoloT182D-mRFP. Insets show magnifications of the outlined regions that highlight KTs with delayed congression to the spindle equator (yellow dashed line). Time 0 is the first shift in KT direction from poleward to anti-poleward movement. (D) Graph represents the variation in SpindlyWT-EGFP levels at KTs highlighted in (C) and its correlation with efficiency of alignment. Spindly-EGFP mean fluorescence intensity (MFI) levels were corrected for photobleaching during acquisition and normalized to the first frame of analysis to account for differences in expression levels. KTs were tracked during the process of congression (using Polo-mRFP signal as a KT reference) and the distance from the unaligned KT pair to the spindle equator was measured. All values were normalized to the last frame of analysis (stable integration in metaphase plate). Time 0 is the first shift in KT direction from poleward to anti-poleward movement (n≥9 KTs from at least 5 cells for each condition, n≥4 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(E) Representative immunofluorescence images of Spindly accumulation at aligned KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP, in control or upon depletion of dynein heavy chain. Insets display magnifications of the outlined regions. CID was used as a KT reference. Data information: Scale bar: 5 μm.(F) Representative immunofluorescence images of Spindly accumulation at unattached KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Cells were treated with colchicine prior to fixation to generate unattached KTs. CID was used as a KT reference. Data information: Scale bar: 5 μm.(G) Graph represents Spindly levels at aligned KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in control PoloWT-EGFP expressing cells, which was set to 100% (n≥633 KTs from at least 35 cells for each condition, n=2 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(H) Graph represents Spindly levels at unattached KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in PoloWT-EGFP expressing cells, which was set to 100% (n≥1282 KTs from at least 59 cells for each condition, n=2 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


Figure 7 ( A ) In vitro kinase assays with the indicated recombinant protein in the presence of [ γ - 32P ] - ATP for 30 min . Phosphorylation was detected by autoradiography and levels of proteins were visualized by coomassie blue staining . Data information : Scale bar : 5 μm . ( D ) Representative immunofluorescence images of Spindly - phospho ( ph ) Ser499 levels at unattached KTs in control Drosophila S2 cells and in Polo - depleted or BI2536 - treated cells . Insets display magnifications of the outlined regions . Cells were treated with colchicine prior to fixation to generate unattached KTs . CID was used as a KT reference . Data information : Scale bar : 5 μm . ( E ) Graph represents the levels of Spindly phS499 at unattached KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity , which was set to 100 % ( n ≥ 1246 KTs from at least 63 cells for each condition , n = 2 independent experiments ) . Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * * * * , < 0 . 0001 Data information : Data are shown as mean ± SD . Scale bar : 5 μm . ( F ) Representative immunofluorescence images of Spindly phS499 levels at unaligned and aligned KTs in a PoloWT - EGFP expressing Drosophila S2 cell . Insets show magnifications of the outlined regions , which highlight either a unaligned ( u ) KT or an aligned ( a ) KT . CID was used as a KT reference . Plotted profiles of signal intensities of phS499 and CID are shown for the highlighted KTs . Data information : Data are shown as mean ± SD . Scale bar : 5 μm . ( G ) Graph represents the levels of Spindly phS499 at unaligned KTs versus aligned KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the mean fluorescence intensity of unaligned KTs , which was set to 100 % ( n ≥ 34 KTs from 14 cells , n = 2 experiments ) . Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * * * , < 0 . 0001 . Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

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 O O O O O O O O O O O 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 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 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

Figure 7(A) In vitro kinase assays with the indicated recombinant protein in the presence of [γ-32P]-ATP for 30 min. Phosphorylation was detected by autoradiography and levels of proteins were visualized by coomassie blue staining. Data information: Scale bar: 5 μm.(D) Representative immunofluorescence images of Spindly-phospho(ph)Ser499 levels at unattached KTs in control Drosophila S2 cells and in Polo-depleted or BI2536-treated cells. Insets display magnifications of the outlined regions. Cells were treated with colchicine prior to fixation to generate unattached KTs. CID was used as a KT reference. Data information: Scale bar: 5 μm.(E) Graph represents the levels of Spindly phS499 at unattached KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥1246 KTs from at least 63 cells for each condition, n=2 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001 Data information: Data are shown as mean ± SD. Scale bar: 5 μm.(F) Representative immunofluorescence images of Spindly phS499 levels at unaligned and aligned KTs in a PoloWT-EGFP expressing Drosophila S2 cell. Insets show magnifications of the outlined regions, which highlight either a unaligned (u) KT or an aligned (a) KT. CID was used as a KT reference. Plotted profiles of signal intensities of phS499 and CID are shown for the highlighted KTs. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.(G) Graph represents the levels of Spindly phS499 at unaligned KTs versus aligned KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the mean fluorescence intensity of unaligned KTs, which was set to 100% (n≥34 KTs from 14 cells, n=2 experiments). Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


Figure 8 ( A ) Western blot analysis of MBP - Zwilch pull downs of full length ( FL ) his6x - SpindlyWT and phosphomimetic mutant his6x - SpindlyS499D . Immobilized MBP was used as negative control . B : Beads , FT : Flow - through . ( B ) Western blot analysis of MBP - Zwilch pull downs of his6x - SpindlyWT or his6x - SpindlyS499D fragments ( aa1 - 510 ) . Immobilized MBP was used as negative control . B : Beads , FT : Flow - through . ( C ) Graph represents the levels of his6x - Spindly relative to MBP - Zwilch from at least three independent pull down experiments . The values obtained for his6x - SpindlyWT were set to 1 . Data information : XXXX Data are shown as mean ± SD . Scale bar : 5 μm . ( D ) Selected frames from live imaging analysis of SpindlyS499A - EGFP or SpindlyS499D - EGFP behavior during KT alignment in Drosophila S2 cells expressing PoloWT - mRFP . SpindlyWT - EGFP expressing cells were used as control . All Spindly - EGFP constructs were resistant to the RNAi targeted against endogenous Spindly . Insets show magnifications of the outlined regions that highlight KTs that delay in congressing to the spindle equator ( yellow dashed line ) . Time 0 is the first shift in KT direction from poleward to anti - poleward movement . ( E ) Graph represents the variation in SpindlyWT - EGFP , SpindlyS499A - EGFP and SpindlyS499D - EGFP levels at KTs highlighted in ( D ) and its correlation with efficiency of alignment . All values were determined as described in Fig . 6D . Time 0 is the first shift in KT direction from poleward to anti - poleward movement ( n ≥ 5 KTs from at least 4 cells for each condition , n ≥ 2 independent experiments ) . Data information : XXXX Data are shown as mean ± SD . Scale bar : 5 μm .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of MBP-Zwilch pull downs of full length (FL) his6x-SpindlyWT and phosphomimetic mutant his6x-SpindlyS499D. Immobilized MBP was used as negative control. B:Beads, FT: Flow-through. (B) Western blot analysis of MBP-Zwilch pull downs of his6x-SpindlyWT or his6x-SpindlyS499D fragments (aa1-510). Immobilized MBP was used as negative control. B:Beads, FT: Flow-through. (C) Graph represents the levels of his6x-Spindly relative to MBP-Zwilch from at least three independent pull down experiments. The values obtained for his6x-SpindlyWT were set to 1. Data information: XXXX Data are shown as mean ± SD. Scale bar: 5 μm.(D) Selected frames from live imaging analysis of SpindlyS499A-EGFP or SpindlyS499D-EGFP behavior during KT alignment in Drosophila S2 cells expressing PoloWT-mRFP. SpindlyWT-EGFP expressing cells were used as control. All Spindly-EGFP constructs were resistant to the RNAi targeted against endogenous Spindly. Insets show magnifications of the outlined regions that highlight KTs that delay in congressing to the spindle equator (yellow dashed line). Time 0 is the first shift in KT direction from poleward to anti-poleward movement. (E) Graph represents the variation in SpindlyWT-EGFP, SpindlyS499A-EGFP and SpindlyS499D-EGFP levels at KTs highlighted in (D) and its correlation with efficiency of alignment. All values were determined as described in Fig.6D. Time 0 is the first shift in KT direction from poleward to anti-poleward movement (n≥5 KTs from at least 4 cells for each condition, n≥2 independent experiments). Data information: XXXX Data are shown as mean ± SD. Scale bar: 5 μm.


Figure 9 ( A ) Representative immunofluorescence images of ZW10 localization to unaligned KTs in Drosophila S2 cells expressing either SpindlyWT - , SpindlyS499A - or SpindlyS499D - EGFP . Insets display magnifications of the outlined regions , which highlight late congressing KTs . CID was used as a KT reference . ( B ) Graph represents the percentage of ZW10 levels at late congressing and non - oriented ( not oriented parallel to the spindle axis ) KTs for cells shown in ( A ) . ZW10 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity , which was set to 100 % ( n ≥ 72 KTs from at least 26 cells for each condition , n = 4 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * , < 0 . 05 ; * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( C ) Selected frames from live imaging analysis of chromosome congression in Drosophila S2 cells expressing SpindlyWT - EGFP , SpindlyS499A - EGFP , SpindlyS499D - EGFP or SpindlyS499D - EGFP in a ZW10 - depleted background . The first frame shows EGFP - positive cells . Chromosome alignment was followed in vivo by direct visualization of KT - associated PoloWT - mRFP signal . Time 0 refers to nuclear envelope breakdown ( NEBD ) , metaphase refers to full KT alignment and Anaphase onset refers to onset of chromosome segregation . ( D ) Quantification of the time spent in prometaphase ( from NEBD until last KT alignment at the metaphase plate ) for cells shown in ( C ) ( n ≥ 10 cells for each condition , n ≥ 2 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * , < 0 . 05 ; * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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 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

Figure 9(A) Representative immunofluorescence images of ZW10 localization to unaligned KTs in Drosophila S2 cells expressing either SpindlyWT-, SpindlyS499A- or SpindlyS499D-EGFP. Insets display magnifications of the outlined regions, which highlight late congressing KTs. CID was used as a KT reference. (B) Graph represents the percentage of ZW10 levels at late congressing and non-oriented (not oriented parallel to the spindle axis) KTs for cells shown in (A). ZW10 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥72 KTs from at least 26 cells for each condition, n=4 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: *, <0.05; **, <0.01; ****, <0.0001 . Data are shown as mean ±SD. Scale bar: 5 μm.(C) Selected frames from live imaging analysis of chromosome congression in Drosophila S2 cells expressing SpindlyWT-EGFP, SpindlyS499A-EGFP, SpindlyS499D-EGFP or SpindlyS499D-EGFP in a ZW10-depleted background. The first frame shows EGFP-positive cells. Chromosome alignment was followed in vivo by direct visualization of KT-associated PoloWT-mRFP signal. Time 0 refers to nuclear envelope breakdown (NEBD), metaphase refers to full KT alignment and Anaphase onset refers to onset of chromosome segregation. (D) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for cells shown in (C) (n≥10 cells for each condition, n≥2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: *, <0.05; **, <0.01; ****, <0.0001 . Data are shown as mean ±SD. Scale bar: 5 μm.


Figure 10 ( A ) Representative immunofluorescence images of ZW10 streaming from KTs in Drosophila S2 cells in metaphase expressing either SpindlyWT - , SpindlyS499A - or SpindlyS499D - EGFP . ZW10 streaming is also shown in S2 cells expressing SpindlyWT - or SpindlyS499A - EGFP in a dynein - depleted background . Insets display magnifications of the outlined regions , which highlight streaming robustness . ( B ) Graph represents the percentage of cells in metaphase showing different levels of ZW10 streaming ( n ≥ 35 cells for each condition , n = 2 independent experiments ) . ( C ) Graph represents the percentage of ZW10 levels at aligned KTs normalized to ZW10 levels at the spindle region . All values were normalized to the mean fluorescence intensity quantified in SpindlyWT - EGFP expressing cells , which was set to 100 % ( n ≥ 470 KTs from at least 35 cells for each condition , n = 2 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm . ( D ) Representative immunofluorescence images of calcium - stable KT - MT attachments in metaphase S2 cells expressing SpindlyWT - , SpindlyS499A - , SpindlyS499D - or SpindlyS499D - EGFP in a ZW10 - depleted background . Insets display magnifications of the outlined regions which highlight different attachment configurations ( E - end on ; L - lateral ; M - merotelic ) . Cartoon depicts the attachment configuration of the respective KT pair . Asterisk highlights an aligned KT pair in which a sister KT appears to be laterally attached to the end of a MT fiber . Plotted profiles show the overlap between CENP - C and tubulin signals for the highlighted KT . CENP - C was used as a KT reference . ( E ) Graph represents the percentage of metaphase cells showing only end - on attachments or at least 1 KT with lateral or merotelic attachment , as shown in D ( n ≥ 44 cells for each condition , n ≥ 2 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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

Figure 10(A) Representative immunofluorescence images of ZW10 streaming from KTs in Drosophila S2 cells in metaphase expressing either SpindlyWT-, SpindlyS499A- or SpindlyS499D-EGFP. ZW10 streaming is also shown in S2 cells expressing SpindlyWT- or SpindlyS499A-EGFP in a dynein-depleted background. Insets display magnifications of the outlined regions, which highlight streaming robustness. (B) Graph represents the percentage of cells in metaphase showing different levels of ZW10 streaming (n≥35 cells for each condition, n=2 independent experiments). (C) Graph represents the percentage of ZW10 levels at aligned KTs normalized to ZW10 levels at the spindle region. All values were normalized to the mean fluorescence intensity quantified in SpindlyWT-EGFP expressing cells, which was set to 100% (n≥470 KTs from at least 35 cells for each condition, n=2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.(D) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing SpindlyWT-, SpindlyS499A-, SpindlyS499D- or SpindlyS499D-EGFP in a ZW10-depleted background. Insets display magnifications of the outlined regions which highlight different attachment configurations (E - end on; L - lateral; M - merotelic). Cartoon depicts the attachment configuration of the respective KT pair. Asterisk highlights an aligned KT pair in which a sister KT appears to be laterally attached to the end of a MT fiber. Plotted profiles show the overlap between CENP-C and tubulin signals for the highlighted KT. CENP-C was used as a KT reference. (E) Graph represents the percentage of metaphase cells showing only end-on attachments or at least 1 KT with lateral or merotelic attachment, as shown in D (n≥44 cells for each condition, n≥2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


Figure 1B Levels of INPP5Eprotein 72 h after transfection of N1E - 115 cells with control ( siControl ) or INPP5E - specific siRNAs ( siINPP5E # 1 or # 2 ) , as determined by immunoblot . C N1E - 115 cells treated with siControl or siINPP5Es were cultured for 2 h in growth medium with or without 125 nM bafilomycin A1 ( Baf . A1 ) , and then analyzed by immunoblot using anti - p62 , anti - LC3 , and anti - GAPDH antibodies . D Quantitation of protein signal intensities from immunoblots in C showing LC3 - II ( left ) or p62 ( right ) levels , following normalization to the control proteinGAPDH . Because Baf . A1 inhibits autophagosome - lysosome fusion and degradation within lysosomes via inhibition of vacuolar - type H + - ATPase , it is possible to measure autophagic flux , i . e . , the degradation of autophagic substrates such as p62 and LC3 - II , by calculating the difference of the signal intensities of these proteins in the presence and absence of Baf . A1 . Results represent the mean ± s . d . of three independent experiments . * * , P < 0 . 01 . E N1E - 115 cells treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - LC3 antibodies and analyzed by immunofluorescencemicroscopy . Bar , 10 µm . F Quantitation of the number of LC3puncta per cell , as described in E ( mean ± s . d . ; n > 100 cells from three independent experiments ) . * * , P < 0 . 01 . G Electron micrographsin N1E - 115 cells treated with siControl or siINPP5Es were cultured in growth medium . Arrowheads indicate autophagosomes . Bar , 1 µm .

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 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 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 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 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

Figure 1B Levels of INPP5Eprotein 72 h after transfection of N1E-115 cells with control (siControl) or INPP5E-specific siRNAs (siINPP5E #1 or #2), as determined by immunoblot.C N1E-115 cells treated with siControl or siINPP5Es were cultured for 2 h in growth medium with or without 125 nM bafilomycin A1 (Baf.A1), and then analyzed by immunoblot using anti-p62, anti-LC3, and anti-GAPDH antibodies.D Quantitation of protein signal intensities from immunoblots in C showing LC3-II (left) or p62 (right) levels, following normalization to the control proteinGAPDH. Because Baf.A1 inhibits autophagosome-lysosome fusion and degradation within lysosomes via inhibition of vacuolar-type H+-ATPase, it is possible to measure autophagic flux, i.e., the degradation of autophagic substrates such as p62 and LC3-II, by calculating the difference of the signal intensities of these proteins in the presence and absence of Baf.A1. Results represent the mean ± s.d. of three independent experiments. **, P < 0.01.E N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LC3 antibodies and analyzed by immunofluorescencemicroscopy. Bar, 10 µm.F Quantitation of the number of LC3puncta per cell, as described in E (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.G Electron micrographsin N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Arrowheads indicate autophagosomes. Bar, 1 µm.