( A ) Representative kymographs showing tetramethylrhodamine ( TMR ) - labeled DDB movement on each type of recombinant MT lattice , as indicated . Scale bars , 5 μm 25 sec . Pink arrowhead marks a diffusive DDB . See also Movie EV1 . ( B ) of DDB velocity on MT lattices lacking each of tubulin - CTTs ( n ≥ 100 DDB complexes per condition from at least three independent experiments ) . No statistical difference was observed between WT mutant MTs ( P ≥ 0 . 1 , unpaired t ) ( C - D ) of number of processive ( C ) , or diffusive ( D ) DDB complexes relative to WT MTs in same chamber ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Processive ( unidirectional motion exceeding 0 . 5 μm ) diffusive ( back - - forth motion ) events were scored in a field of view then divided by lengths of MTs time , thus normalizing to a value of number of motility events per μm MT per sec . Error bars SD .

O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O 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-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

(A) Representative kymographs showing tetramethylrhodamine (TMR)-labeled DDB movement on each type of recombinant MT lattice, as indicated. Scale bars, 5 μm and 25 sec. Pink arrowhead marks a diffusive DDB complex. See also Movie EV1. (B) Quantification of DDB velocity on MT lattices lacking each of the tubulin-CTTs (n ≥ 100 DDB complexes per condition from at least three independent experiments). No statistical difference was observed between WT and mutant MTs (P ≥ 0.1, unpaired t test) (C-D) Quantification of the number of processive (C), or diffusive (D) DDB complexes relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Processive (unidirectional motion exceeding 0.5 μm) and diffusive (back-and-forth motion) events were scored in a field of view and then divided by the lengths of MTs and time, thus normalizing to a value of number of motility events per μm MT per sec. Error bars SD.


( E - F ) TIRF of 1 nM TMR - labeled GST - hDyn ( 2 mM ATP ) ( E ) , or 0 . 3 nM TMR - labeled p150 ( F ) molecules ( green ) bound to either Δα - CTT ( top ) , or Δβ - CTT MTs ( bottom ) . Pink arrows show mutant MTs in both raw , SD map , highlighting differences in binding between WT mutant MTs that are apparent in SD map . Right , standard deviation maps from an entire time reveal ensemble binding dissociation events that lead to variations in pixel intensities ( see Methods ) . Scale bars are 5 μm . ( G ) of mean ( arbitrary units ) per μm MT for GST - hDyn ( 2 mM ATP ) or p150 bound to indicated mutant MT relative to WT MTs in same chamber . Image shows first frame from time series . of GST - hDyn was quantified from maximum projections of entire time due to transient binding of GST - hDyn to MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD . P value ( unpaired t ) of Δα - CTT vs . Δβ - CTT MTs = 0 . 5208 for GST - hDyn , ≤ 0 . 0001 for p150 .

O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O 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-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O

(E-F) TIRF images of 1 nM TMR-labeled GST-hDyn (2 mM ATP) (E), or 0.3 nM TMR-labeled p150 (F) molecules (green) bound to either Δα-CTT (top), or Δβ-CTT MTs (bottom). Pink arrows show mutant MTs in both the raw, and SD map images, highlighting differences in binding between WT and mutant MTs that are apparent in the SD map. Right, standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities (see Methods). Scale bars are 5 μm. (G) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. Image shows first frame from time series. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD. P value (unpaired t test) of Δα-CTT vs. Δβ-CTT MTs = 0.5208 for GST-hDyn, and ≤ 0.0001 for p150.


( A ) TIRF image of TMR - labeled DDB molecules ( green ) bound to either tyrosinated ( red ) or detyrosinated ( blue ) MTs . A standard deviation map of whole time is shown , revealing preferential association of DDB with tyrosinated MTs versus detyrosinated MTs ( pink arrow ) . Representative kymographs of DDB movement are shown from each type of MT present in same chamber . Scale bars are 5 μm 25 sec . See also Movie EV2 . ( B - C ) of number of processive ( B ) or diffusive ( C ) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in same chamber ( n ≥ 20 MTs quantified for each condition from three independent experiments ) . Error bars represent SD . Data from Δα - CTT MTs ( Fig . 1 ) are re - plotted for comparison . ( D ) of frequency of processive DDB molecules as a function of amount of tyrosinated tubulin incorporated into lattice normalized to fully tyrosinated MTs . Data points from two independent experiments are shown .

O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O 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-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O

(A) TIRF image of TMR-labeled DDB molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. A standard deviation map of the whole time sequence is shown, revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs (pink arrow). Representative kymographs of DDB movement are shown from each type of MT present in the same chamber. Scale bars are 5 μm and 25 sec. See also Movie EV2. (B-C) Quantification of the number of processive (B) or diffusive (C) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber (n ≥ 20 MTs quantified for each condition from three independent experiments). Error bars represent SD. Data from Δα-CTT MTs (Fig. 1) are re-plotted for comparison. (D) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs. Data points from two independent experiments are shown.


( E ) TIRF of 0 . 3 nM TMR - labeled p150 ( top ) or 1 nM TMR - labeled GST - hDyn ( bottom ) molecules ( green ) bound to either tyrosinated ( red ) or detyrosinated ( blue ) MTs . Right , standard deviation maps of binding over an entire time kymographs from either tyrosinated or detyrosinated MTs are shown . Scale bars are 5 μm 25 sec . ( F ) of mean ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2 mM ATP ) or p150 bound to indicated mutant MT relative to WT MTs in same chamber . of GST - hDyn was quantified from maximum projections of entire time due to transient binding of GST - hDyn to MT . ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(E) TIRF images of 0.3 nM TMR-labeled p150 (top) or 1 nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown. Scale bars are 5 μm and 25 sec. (F) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT. (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.


( A ) Immunoblot of brain tubulin with or without carboxypeptidase A ( CPA ) . Note CPA specifically removes α - tubulin C - terminal tyrosine without affecting other PTMs .

O O O B-EXP_ASSAY O B-TISSUE B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(A) Immunoblot of brain tubulin with or without carboxypeptidase A (CPA). Note CPA specifically removes the α-tubulin C-terminal tyrosine without affecting other PTMs.


( B ) TIRF image of TMR - labeled DDB molecules ( green ) on either WT ( red ) or CPA - treated ( blue ) pig brain MTs . Standard deviation map of entire time shows DDB preference for WT MTs kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm 25 sec . ( C ) of number of processive DDB complexes per μm MT per sec on CPA - treated MTs , relative to WT MTs in same chamber ( n ≥ 7 MTs of each type quantified from at least three independent experiments ) . Error bars represent SD .

O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM B-TISSUE B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(B) TIRF image of TMR-labeled DDB molecules (green) on either WT (red) or CPA-treated (blue) pig brain MTs. Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec. (C) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA-treated MTs, relative to WT MTs in the same chamber (n ≥ 7 MTs of each type quantified from at least three independent experiments). Error bars represent SD.


( D ) TIRF of 0 . 3nM TMR - labeled p150 ( top ) or 1nM TMR - labeled GST - hDyn ( bottom ) molecules ( green ) bound to either WT ( red ) , or CPA - treated MTs ( blue ) . Right , standard deviation maps of binding over an entire time kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm 25 sec for p150 , 5 μm 12 sec for GST - hDyn ( E ) of mean ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2mM ATP ) or p150 bound to CPA - treated MTs relative to WT MTs in same chamber . of GST - hDyn was quantified from maximum projections of entire time due to transient binding of GST - hDyn to MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(D) TIRF images of 0.3nM TMR-labeled p150 (top) or 1nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either WT (red), or CPA-treated MTs (blue). Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec for p150, and 5 μm and 12 sec for GST-hDyn (E) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2mM ATP) or p150 bound to CPA-treated MTs relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.


( A ) Schematic of p150 constructs used . p135 isoform lacks CAP - Gly domain , but retains part of adjacent basic domain ( red ) coiled - coil domains ( blue ) . Right , Coomassie blue stained gel of purified p150 p135 . Molecular markers are indicated .

O O O O O B-GENEPROD O O O B-GENEPROD O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O O

(A) Schematic of the p150 constructs used. The p135 isoform lacks the CAP-Gly domain, but retains part of the adjacent basic domain (red) and coiled-coil domains (blue). Right, Coomassie blue stained gel of the purified p150 and p135 proteins. Molecular weight markers are indicated.


( B ) TIRF image of either p150 ( 0 . 3 nM ) , or p135 ( 0 . 3 or 6 nM ) bound to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Note : no binding of p135 is observed at comparable concentrations to p150 indicating lower binding affinity of this isoform . were visualized by from GFP tag fused to each construct .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O

(B) TIRF image of either p150 (0.3 nM), or p135 (0.3 or 6 nM) bound to either WT (red), or CPA-treated porcine MTs (blue). Note: no binding of p135 is observed at comparable concentrations to p150 indicating the lower binding affinity of this isoform. Proteins were visualized by fluorescence from the GFP tag fused to each construct.


( C ) Still from a TIRF time - series of p135 ( green ) binding to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Right , standard deviation map of p135 binding shows no preference for either type of MT , of mean ( arbitrary units ) per μm MT for p135 bound to CPA - treated MTs relative to WT MTs in same chamber ( n ≥ 10 MTs quantified for each condition from three independent experiments ) . Scale bars are 5 μm .

O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

(C) Still images from a TIRF time-series of p135 (green) binding to either WT (red), or CPA-treated porcine MTs (blue). Right, standard deviation map of p135 binding shows no preference for either type of MT, and quantification of mean fluorescence intensity (arbitrary units) per μm MT for p135 bound to CPA-treated MTs relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from three independent experiments). Scale bars are 5 μm.


( B ) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated ( red ) detyrosinated ( blue ) sections . Most TMR - labeled DDB molecules ( green ) move processively through annealed junction between two types of MT lattice ( 65 % , n = 836 processive complexes , 65 MT - MT junctions , three independent experiments ) . Diffusive DDB molecules ( arrow ) are primarily observed on tyrosinated section of MT do not diffuse across boundary . Right , video frames showing a processive DDB ( pink arrowhead ) moving across junction ( yellow arrowhead ) of tyrosinated detryosinated MT . ( + ) ( - ) signs denote MT polarity inferred from directionality of DDB movement . Scale bars are 5 μm 25 sec . See also Movie EV3 .

O O O O O O O B-SUBCELLULAR O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

(B) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated (red) and detyrosinated (blue) sections. Most TMR-labeled DDB molecules (green) move processively through the annealed junction between the two types of MT lattice (65%, n=836 processive complexes, 65 MT-MT junctions, three independent experiments). Diffusive DDB molecules (white arrow) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary. Right, video frames showing a processive DDB complex (pink arrowhead) moving across the junction (yellow arrowhead) of tyrosinated and detryosinated MT. The (+) and (-) signs denote MT polarity inferred from the directionality of DDB movement. Scale bars are 5 μm and 25 sec. See also Movie EV3.


( C ) TIRF of low ( 0 . 3 nM ) high ( 10 nM ) concentrations of TMR - labeled p150 ( green ) bound to chimeric yeast MTs containing tyrosinated ( red ) detyrosinated ( blue ) sections of MT . Representative kymographs below show diffusive behavior of p150 molecules on MT . Note that p150 rarely diffuses into detyrosinated section of MT , even at high concentrations . Scale bars are 5 μm 10 sec .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-ORGANISM B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

(C) TIRF images of low (0.3 nM) and high (10 nM) concentrations of TMR-labeled p150 (green) bound to chimeric yeast MTs containing tyrosinated (red) and detyrosinated (blue) sections of MT. Representative kymographs below show diffusive behavior of p150 molecules on the MT. Note that p150 rarely diffuses into the detyrosinated section of MT, even at high concentrations. Scale bars are 5 μm and 10 sec.


( D - E ) TIRFimages kymographs of annealed WT CPA - treated porcineMTs ( D ) , or annealed WT MTs ( E ) . majority of DDB complexes ( 78 % , n = 960 processive complexes , 45 MT - MT junctions , three independent experiments ) move processively across boundary between WT CPA - MTs ( D ) . Note TMR - labeled DDB complexes ( green ) in ( E ) traverse annealed boundary without pausing ( 93 % , n = 677 processive complexes , 45 MT - MT junctions , three independent experiments ) , suggesting that annealed junction does not affect DDB motility .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-ORGANISM O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O

(D-E) TIRFimages and kymographs of annealed WT and CPA-treated porcineMTs(D), or annealed WT MTs(E). The majority of DDB complexes (78%, n=960 processive complexes, 45 MT-MT junctions, three independent experiments) move processively across the boundary between WT and CPA-MTs(D). Note TMR-labeled DDB complexes (green) in (E) traverse the annealed boundary without pausing (93%, n=677 processive complexes, 45 MT-MT junctions, three independent experiments), suggesting that the annealed junction does not affect DDB motility.


( F ) TIRF image kymograph from a chimeric MT composed of WT ( red ) subtilisin - treated porcine MTs ( blue ) . Note some DDB complexes ( pink arrows ) traverse boundary continue moving along subtilisin - treated section of MT ( 41 % , n = 709 processive complexes , 56 MT - MT junctions , 3 independent experiments ) . scale bars in D - F are 5 μm 25 sec .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O B-ORGANISM B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

(F) TIRF image and kymograph from a chimeric MT composed of WT (red) and subtilisin-treated porcine MTs (blue). Note some DDB complexes (pink arrows) traverse the boundary and continue moving along the subtilisin-treated section of the MT (41%, n=709 processive complexes, 56 MT-MT junctions, 3 independent experiments). All scale bars in D-F are 5 μm and 25 sec.


( A ) UASp - GFP - mCherry - DrAtg8a / + ; nosGAL4 / + flies conditioned on yeast paste had diffuse GFP - mCherry - DrAtg8a in midstage egg chambers . ( B ) Nondegenerating midstage egg chambers from starved flies contained autophagosomes ( yellow ) autolysosomes ( red ) . ( C D ) Egg chambers early in degeneration process showed follicle that take up portions of nurse cytoplasm ( C ) followed by condensation fragmentation of nurse nuclei further uptake of nurse cytoplasm into follicle ( D ) . ( E ) Late stage degenerating egg chambers lose GFP fluoresce red .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O B-TISSUE I-TISSUE O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O B-CELL_TYPE B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O

(A) UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies conditioned on yeast paste had diffuse GFP-mCherry-DrAtg8a staining in midstage egg chambers. (B) Nondegenerating midstage egg chambers from starved flies contained autophagosomes (yellow) and autolysosomes (red). (C and D) Egg chambers early in the degeneration process showed follicle cells that take up portions of the nurse cell cytoplasm (C) followed by condensation and fragmentation of the nurse cell nuclei and further uptake of the nurse cell cytoplasm into follicle cells (D). (E) Late stage degenerating egg chambers lose all GFP staining and fluoresce red.


( F ) Fed Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies showed diffuse GFP - mCherry - DrAtg8a in germline . ( G ) Starved Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers . ( H I ) Fed flies overexpressing truncated Dcp - 1 ( tDcp - 1 ) in germline showed increased autophagosomes autolysosomes in nondegenerating midstage egg chambers ( H ) also contained degenerating midstage egg chambers that lose GFP fluoresce red ( I ) . Bars : ( main ) 25 µm ; ( insets ) 12 . 5 µm . Insets in A - I show diffuse of Atg8a or autophagosomes autolysosomes . ( J K ) percentages of autolysosomes ( autolysosomes / total autophagic structures ) were manually calculated in at least eight egg chambers for each genotype as indicated . Error bars represent means ± SD . Statistical testing was performed using one - way ANOVA with a Dunnet post ( * * * * , P < 0 . 0001 ) or a two - Student ' s t ( * * , P < 0 . 005 ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O B-SUBCELLULAR O O O B-TISSUE I-TISSUE O O O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O O O O O 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-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Fed Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed diffuse GFP-mCherry-DrAtg8a staining in the germline. (G) Starved Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers. (H and I) Fed flies overexpressing truncated Dcp-1 (tDcp-1) in the germline showed increased autophagosomes and autolysosomes in nondegenerating midstage egg chambers (H) and also contained degenerating midstage egg chambers that lose all GFP fluorescence and fluoresce red (I). Bars: (main images) 25 µm; (insets) 12.5 µm. Insets in A-I show diffuse cytoplasmic staining of Atg8a or autophagosomes and autolysosomes. (J and K) The percentages of autolysosomes (autolysosomes/total autophagic structures) were manually calculated in at least eight egg chambers for each genotype as indicated. Error bars represent the means ± SD. Statistical testing was performed using one-way ANOVA with a Dunnet post test (****, P < 0.0001) or a two-tailed Student's t test (**, P < 0.005).


( A ) Drosophila S2 stably expressing GFP - RFP - Atg8a showed an increase in of containing two or more autolysosomes after starvation , which was blocked after Bafilomycin A1 ( BafA1 ) treatment . At least 50 were manually quantitated in three independent experiments ( n = 3 ) . Statistical significance was determined using one - way ANOVA with a Dunnett post ( * * , P < 0 . 01 ) . ( B ) Representative of S2 - GFP - RFP - Atg8a after indicated treatments .

O O O B-ORGANISM B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

(A) Drosophila S2 cells stably expressing GFP-RFP-Atg8a showed an increase in the percentage of cells containing two or more autolysosomes after starvation, which was blocked after Bafilomycin A1 (BafA1) treatment. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01). (B) Representative images of S2-GFP-RFP-Atg8a cells after the indicated treatments.


( C ) S2 - GFP - RFP - Atg8a were treated with dsRNAs subjected to fed or starvation conditions as indicated . At least 50 were manually quantitated in three independent experiments ( n = 3 ) . Statistical significance was determined using one - way ANOVA with a Dunnett post ( * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ) . ( D ) Representative of S2 - GFP - RFP - Atg8a after indicated RNAi treatments . Error bars represent means ± SD . Bars , 10 µm .

O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

(C) S2-GFP-RFP-Atg8a cells were treated with dsRNAs and subjected to fed or starvation conditions as indicated. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D) Representative images of S2-GFP-RFP-Atg8a cells after the indicated RNAi treatments. Error bars represent the means ± SD. Bars, 10 µm.


( A ) Nondegenerating midstage egg chambers from fed w1118flies showed Ref ( 2 ) P in follicle nurse . ( B ) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref ( 2 ) P . ( D ) Starved Atg7flies showed an accumulation of Ref ( 2 ) P in follicle nurse of degenerating midstage egg chambers . ( E ) Starved Dcp - 1Prev1flies contained increased of Ref ( 2 ) P in degenerating midstage egg chambers .

O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O

(A) Nondegenerating midstage egg chambers from fed w1118flies showed Ref(2)P staining in follicle cells and nurse cells. (B) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref(2)P.(D) Starved Atg7flies showed an accumulation of Ref(2)P in the follicle cells and nurse cells of degenerating midstage egg chambers. (E) Starved Dcp-1Prev1flies contained increased levels of Ref(2)P in degenerating midstage egg chambers.


( C ) A representative Western blot of ovaries from w1118 Atg7d77 / d14 flies subjected to fed or starvation conditions for 4 d . Ref ( 2 ) P was detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate Ref ( 2 ) P relative to actin . Graph represents ± SD from five independent experiments ( n = 5 ) . Statistical significance was determined using a two - Student ' s t . * , P = 0 . 004 for fed samples ; * , P = 0 . 008 for starved samples .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-GENEPROD O O B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) A representative Western blot of ovaries from w1118 and Atg7d77/d14 flies subjected to fed or starvation conditions for 4 d. Ref(2)P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2)P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test. *, P = 0.004 for fed samples; *, P = 0.008 for starved samples.


( F ) A representative Western blot of ovaries from w1118 Dcp - 1Prev1 flies that were subjected to fed or starvation conditions for 4 d . Ref ( 2 ) P was detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate Ref ( 2 ) P relative to actin . Graph represents ± SD from five independent experiments ( n = 5 )

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O

(F) A representative Western blot of ovaries from w1118 and Dcp-1Prev1 flies that were subjected to fed or starvation conditions for 4 d. Ref(2)P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2)P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5)


( A B ) l ( 2 ) mbn were labeled with antibodies to Dcp - 1 ATPsyn - α ( A ) or MitoTracker red ( MTR ; B ) . Merged show colocalization between Dcp - 1 mitochondria . Boxes represent zoomed . Bars : ( main ) 5 µm ; ( zoomed ) 1 . 25 µm .

O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

(A and B) l(2)mbn cells were labeled with antibodies to Dcp-1 and ATPsyn-α (A) or MitoTracker red (MTR; B). Merged images show colocalization between Dcp-1 and the mitochondria. Boxes represent zoomed images. Bars: (main images) 5 µm; (zoomed images) 1.25 µm.


( C ) Western blot from l ( 2 ) mbn subjected to nutrient - rich or starvation conditions for 6 h . were separated into ( C ) mitochondrial enriched ( M ) fractions .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O

(C) Western blot from l(2)mbn cells subjected to nutrient-rich or starvation conditions for 6 h. Cells were separated into cytosolic (C) and mitochondrial enriched (M) fractions.


( D ) Ovaries from fed w1118 flies were separated into - enriched fractions probed with antibodies to VDAC , Tubulin , ATPsyn - α , Dcp - 1 .

O O O B-TISSUE O O B-ORGANISM I-ORGANISM O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

(D) Ovaries from fed w1118 flies were separated into cytosolic and membrane-enriched fractions and probed with antibodies to VDAC, Tubulin, ATPsyn-α, and Dcp-1.


( E F ) Intact lysed mitochondria ( E ) or intact mitochondria isolated from l ( 2 ) mbn ( F ) were treated with proteinase K ( PK ) . effects of proteinase K treatment were assessed by antibodies to VDAC , ATPsyn - α , Pink1 , Dcp - 1 .

O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

(E and F) Intact and lysed mitochondria (E) or intact mitochondria isolated from l(2)mbn cells (F) were treated with proteinase K (PK). The effects of proteinase K treatment were assessed by antibodies to VDAC, ATPsyn-α, Pink1, and Dcp-1.


O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O


( G ) Control Dcp - 1 RNAi - treated were subjected to nutrient - rich or starvation conditions stained with NAO . Mean was measured by flow cytometry . Graph represents ± of three independent experiments ( n = 3 ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

(G) Control and Dcp-1 RNAi-treated cells were subjected to nutrient-rich or starvation conditions and stained with NAO. Mean fluorescence was measured by flow cytometry. Graph represents ± SEM of three independent experiments (n = 3).


( A ) l ( 2 ) mbn were labeled with ATPsyn - α , mitochondrial was scored as fragmented , normal , or elongated . ( B ) were treated with control or Dcp - 1 dsRNA subjected to nutrient - rich media or 1 h of starvation . Quantifications represent of with elongated mitochondria divided by total number of examined . At least 100 were examined manually in three independent experiments ( n = 3 ) . Error bars represent mean ± SD . Statistical significance was determined using one - way ANOVA with a Bonferroni post ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) l(2)mbn cells were labeled with the ATPsyn-α, and mitochondrial morphology was scored as fragmented, normal, or elongated. (B) Cells were treated with control or Dcp-1 dsRNA and subjected to nutrient-rich media or 1 h of starvation. Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined. At least 100 cells were examined manually in three independent experiments (n = 3). Error bars represent the mean ± SD. Statistical significance was determined using one-way ANOVA with a Bonferroni post test (*, P < 0.05; **, P < 0.01).


( C ) Mitochondrial targeted GFP ( mitoGFP ) was expressed in germline using nosGAL4 driver . shows mitoGFP , Armadillo , . ( D ) Mitochondria were scored as healthy ( H ) , clustered ( C ) , or elongated overly connected ( E ) . of mitochondria from fed UASp - mitoGFP / + ; nosGAL4 / + flies were scored as healthy . n = 15 egg chambers manually scored . ( E ) mitoGFP was expressed in Dcp - 1Prev1 flies using nosGAL4 driver . ( F ) 54 % of egg chambers from UASp - mitoGFP / + ; Dcp - 1Prev1 / Dcp - 1Prev1 ; nosGAL4 / + flies contained elongated mitochondria , 39 % contained mitochondria that were scored as healthy , 7 % contained clustered mitochondria . n = 28 egg chambers manually scored .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O O

(C) Mitochondrial targeted GFP (mitoGFP) was expressed in the germline using the nosGAL4 driver. Staining shows mitoGFP, Armadillo, and DNA. (D) Mitochondria were scored as healthy (H), clustered (C), or elongated and overly connected (E). All of mitochondria from fed UASp-mitoGFP/+;nosGAL4/+ flies were scored as healthy. n = 15 egg chambers manually scored. (E) mitoGFP was expressed in Dcp-1Prev1 flies using the nosGAL4 driver. (F) 54% of egg chambers from UASp-mitoGFP/+;Dcp-1Prev1/Dcp-1Prev1;nosGAL4/+ flies contained elongated mitochondria, 39% contained mitochondria that were scored as healthy, and 7% contained clustered mitochondria. n = 28 egg chambers manually scored.


( A ) Total ATP were measured in l ( 2 ) mbn treated with control or Dcp - 1 dsRNA . Data represent ± of three independent experiments ( n = 3 ) . Statistical significance was determined using a two - Students t ( * , P = 0 . 02 ) .

O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Total cellular ATP levels were measured in l(2)mbn cells treated with control or Dcp-1 dsRNA. Data represent ± SEM of three independent experiments (n = 3). Statistical significance was determined using a two-tailed Students t test (*, P = 0.02).


( B ) Total ATP were measured in ovaries from fed or starved w1118 Dcp - 1Prev1 flies . Data represent ± of five independent experiments ( n = 5 ) . Statistical significance was determined using a two - Student ' s t ( * * , P = 0 . 014 ; * * * , P < 0 . 001 ) .

O O O O B-SMALL_MOLECULE O O O B-TISSUE O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O 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) Total cellular ATP levels were measured in ovaries from fed or starved w1118 and Dcp-1Prev1 flies. Data represent ± SEM of five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test (**, P = 0.014; ***, P < 0.001).


( C ) Fed or starved w1118 Dcp - 1Prev1 flies were treated with DMSO or 25 µg / ml oligomycin A , Ref ( 2 ) P were assessed by immunoblot . Actin served as a loading control . Densitometry was performed to quantitate relative to actin . Graph represents ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - Student ' s t ( * , P = 0 . 02 ) .

O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Fed or starved w1118 and Dcp-1Prev1 flies were treated with DMSO or 25 µg/ml oligomycin A, and Ref(2)P levels were assessed by immunoblot analysis. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graph represents ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P = 0.02).


( D E ) Control UASp - GFP - mCherry - DrAtg8a / + ; nosGAL4 / + flies ( D ) Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies ( E ) were subjected to starvation conditions supplemented with DMSO or 25 µg / ml oligomycin A . Bars , 25 µm . Quantifications show of autolysosomes ( autolysosomes / total autophagic structures ) . At least eight egg chambers were manually quantitated per genotype per condition ( n = 8 ) . Statistical testing was determined using a two - Student ' s t . * * * , P = 0 . 0002 .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D and E) Control UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies (D) and Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies (E) were subjected to starvation conditions supplemented with DMSO or 25 µg/ml oligomycin A. Bars, 25 µm. Quantifications show percentage of autolysosomes (autolysosomes/total autophagic structures). At least eight egg chambers were manually quantitated per genotype per condition (n = 8). Statistical testing was determined using a two-tailed Student's t test. ***, P = 0.0002.


( A ) A representative Western blot showing SesB ATPsyn - α from fed or starved l ( 2 ) mbn treated with control or Dcp - 1 RNAi . Actin served as a loading control . Densitometry was performed to quantitate relative to actin . Graphs represent ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - Student ' s t ( * , P < 0 . 05 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) A representative Western blot showing SesB and ATPsyn-α levels from fed or starved l(2)mbn cells treated with control or Dcp-1 RNAi. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P < 0.05).


( B ) A representative Western blot of ovaries from fed or starved w1118 or Dcp - 1Prev1 flies . SesB ATPsyn - α were detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate relative to actin . Graphs represent ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - Student ' s t ( * , P < 0 . 05 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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) A representative Western blot of ovaries from fed or starved w1118 or Dcp-1Prev1 flies. SesB and ATPsyn-α were detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P < 0.05).


( C ) Purified catalytically active Dcp - 1FL , but not catalytically inactive Dcp - 1C < A , in vitro translated Drice . Cleavage of in vitro translated SesB was not detected .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O

(C) Purified catalytically active Dcp-1FL, but not catalytically inactive Dcp-1C<A, cleaved in vitro translated Drice. Cleavage of in vitro translated SesB was not detected.


( D ) V5 - tagged Dcp - 1C < A or V5 vector only control was expressed in l ( 2 ) mbn subjected to nutrient - full or starvation conditions for 2 h . After IP with anti - V5 antibodies , lysates were separated by SDS - PAGE . were visualized with colloidal Coomassie stain .

O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(D) V5-tagged Dcp-1C<A or V5 vector only control was expressed in l(2)mbn cells and subjected to nutrient-full or starvation conditions for 2 h. After IP with anti-V5 antibodies, lysates were separated by SDS-PAGE. Proteins were visualized with colloidal Coomassie stain.


( E ) FLAG - SesB or a vector only control was expressed in l ( 2 ) mbn was immunoprecipitated with FLAG - agarose . Immunoblots show interaction between FLAG - SesB endogenous pro - Dcp - 1 . asterisk represents a nonspecific band .

O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(E) FLAG-SesB or a vector only control was expressed in l(2)mbn cells and was immunoprecipitated with FLAG-agarose. Immunoblots show the interaction between FLAG-SesB and endogenous pro-Dcp-1. The asterisk represents a nonspecific band.


( A ) Ovaries from fed SesBOrg flies show reduced Ref ( 2 ) P in degenerating midstage egg chamber ( arrow ) .

O O O B-TISSUE O O B-GENEPROD B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O

(A) Ovaries from fed SesBOrg flies show reduced Ref(2)P staining in degenerating midstage egg chamber (arrow).


( B ) Ovaries from fed w1118 SesBOrg were analyzed by Western blotting using Ref ( 2 ) P . Actin served as a loading control . Results are representative of three independent experiments ( n = 3 ) .

O O O B-TISSUE O O B-ORGANISM B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(B) Ovaries from fed w1118 and SesBOrg were analyzed by Western blotting using Ref(2)P. Actin served as a loading control. Results are representative of three independent experiments (n = 3).


( C ) SesBOrg flies expressing GFP - mCherry - DrAtg8a in germline showed increased autophagosomes autolysosomes in nondegenerating ( left ) degenerating ( right ) midstage egg chambers . Insets show autophagosomes autolysosomes ( left ) autolysosomes ( right ) in SesBOrg midstage egg chambers .

O O O B-GENEPROD B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-TISSUE I-TISSUE O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-GENEPROD O B-TISSUE I-TISSUE O

(C) SesBOrg flies expressing GFP-mCherry-DrAtg8a in the germline showed increased autophagosomes and autolysosomes in nondegenerating (left) and degenerating (right) midstage egg chambers. Insets show autophagosomes and autolysosomes (left) and autolysosomes (right) in SesBOrg midstage egg chambers.


( D ) Quantitation of percentages of autolysosomes ( autolysosomes / total autophagic structures ) in nondegenerating ovaries from fed w1118 SesBOrg flies . 10 egg chambers were manually examined per genotype . Error bars represent means ± SD . Statistical significance was determined using a two - Student ' s t ( * * * , P < 0 . 0001 ) .

O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-TISSUE O O B-ORGANISM B-GENEPROD B-ORGANISM O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitation of percentages of autolysosomes (autolysosomes/total autophagic structures) in nondegenerating ovaries from fed w1118 and SesBOrg flies. 10 egg chambers were manually examined per genotype. Error bars represent the means ± SD. Statistical significance was determined using a two-tailed Student's t test (***, P < 0.0001).


( E ) Degenerating midstage egg chambers ( arrows ) from fed SesBOrg flies are TUNEL positive .

O O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD B-ORGANISM O B-EXP_ASSAY O O

(E) Degenerating midstage egg chambers (arrows) from fed SesBOrg flies are TUNEL positive.


( F ) Degenerating midstage egg chambers from starved control SesBOrg / FM7a ; Dcp - 1Prev1 / Dcp - 1Prev1flies contained persisting nurse cellnuclei increased Ref ( 2 ) P . n = 117 ovarioles examined . Box represents zoomed image . ( G ) Degenerating midstage egg chambers from fed SesBOrg ; Dcp - 1Prev1flies contained condensed fragmented nurse cellnuclei low of Ref ( 2 ) P . Box represents zoomed image . n = 156 ovarioles examined .

O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-TISSUE O O

(F) Degenerating midstage egg chambers from starved control SesBOrg/FM7a;Dcp-1Prev1/Dcp-1Prev1flies contained persisting nurse cellnuclei and increased Ref(2)P. n = 117 ovarioles examined. Box represents zoomed image. (G) Degenerating midstage egg chambers from fed SesBOrg;Dcp-1Prev1flies contained condensed and fragmented nurse cellnuclei and low levels of Ref(2)P. Box represents zoomed image. n = 156 ovarioles examined.


( B ) Immunoblotting of brown preadipocytes ( WT - 1 ) stimulated with insulin at different doses for 5 min ( n = 3 ) . molecular corresponded to I - Afadin isoform .

O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O

(B) Immunoblotting and quantification of brown preadipocytes (WT-1) stimulated with insulin at different doses for 5 min (n=3). The molecular weight corresponded to the I-Afadin isoform.


( C ) Immunoblotting of mature brown adipocytes ( WT - 1 ) stimulated with insulin ( 100nM ) for 5 min ( n = 4 ) .

O O O B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(C) Immunoblotting of mature brown adipocytes (WT-1) stimulated with insulin (100nM) for 5 min (n=4).


( D ) Immunoblotting of immortalized human mature brown adipocytes stimulated with insulin for 5 min ( n = 3 ) .

O O O B-EXP_ASSAY O O B-ORGANISM B-TISSUE I-TISSUE I-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O

(D) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min (n=3).


( E ) Immunoblotting of human mature adipocytes stimulated with insulin for 5 min ( n = 3 ) .

O O O B-EXP_ASSAY O B-ORGANISM B-TISSUE I-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O

(E) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min (n=3).


( F ) Immunoblotting of isolated brown adipose tissue ( BAT ) from C57BL / 6J mice injected with 5U insulin for 5 min ( n = 4 - 6 ) .

O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O

(F) Immunoblotting and quantification of isolated brown adipose tissue (BAT) from male C57BL/6J mice injected with 5U insulin for 5 min (n=4-6).


( G ) of immunoblotting of isolated BAT , subcutaneous adipose tissue ( sWAT ) visceral adipose tissue ( vWAT ) from C57BL / 6J mice after fasting or re - feeding for 2 / 6 hrs ( n = 6 ) .

O O O O B-EXP_ASSAY O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

(G) Quantification of immunoblotting of isolated BAT, subcutaneous white adipose tissue (sWAT) and visceral white adipose tissue (vWAT) from male C57BL/6J mice after fasting or re-feeding for 2/6 hrs (n=6).


( H ) Immunoblotting of total Afadin S1795 phosphorylation in adipose tissue depots from 12 - week old C57BL / 6 mice fed with either chow ( CD ) or high - fat diet ( HFD ) for 6 - injected with vehicle or insulin ( n = 6 ) .

O O O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

(H) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12-week old C57BL/6 male mice fed with either chow (CD) or high-fat diet (HFD) for 6-weeks injected with vehicle or insulin (n=6).


( I ) Immunoblotting of Afadin S1718 phosphorylation in human Stromal vascular fractions ( SVF ) from lean , obese or Type 2 Diabetic ( T2DM ) subjects stimulated or not with insulin ( n = 6 ) . Representative Western blots are shown . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-ORGANISM B-TISSUE I-TISSUE O O B-TISSUE O O O O B-DISEASE O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions (SVF) from lean, obese or Type 2 Diabetic (T2DM) subjects stimulated or not with insulin (n=6). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . BAT ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-TISSUE O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O O O O O

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . BAT of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . sWAT ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . sWAT ; of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-TISSUE O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . Liver ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . Liver of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . ( G ) Quantifications of phosphorylated Insulin Receptor β phosphorylated Akt across three tissues ( n = 4 - 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (G) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . ( H ) Quantifications of Total IRβ / tubulin in BAT , sWAT Liver ( n = 5 - 6 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (H) Quantifications of Total IRβ/tubulin in BAT, sWAT and Liver (n=5-6). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


lysates from 10 - 13 - week old CTRL FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted quantified . ( I ) Insulin - induced lipogenesis in isolated mature adipocytes from CTRL FAKO ( Mllt4 - / - ) mice ( n = 5 ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (I) Insulin-induced lipogenesis in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( A ) CRISPR / nCas9 - mediated ablation of Afadin in a clonal population termed Afdn - KO .

O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O

(A) CRISPR/nCas9-mediated ablation of Afadin protein levels in a clonal cell population termed Afdn-KO.


( B ) Oil red O gene expression of selected adipogenic genes in WT - 1 Afdn - KO differentiated adipocytes ( n = 3 ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oil red O staining and gene expression of selected adipogenic genes in WT-1 and Afdn-KO differentiated adipocytes (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( C ) Wild - type , Afdn - KO Afdn - KO with re - expression FLAG - tagged WT I - Afadin or a non - phosphorylable form ( S1795A ) stimulated with vehicle or insulin ( n = 4 ) for Immunoblotting .

O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O

(C) Wild-type, Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT I-Afadin or a non-phosphorylable form (S1795A) stimulated with vehicle or insulin (n=4) for Immunoblotting.


( D ) of insulin - induced Akt phosphorylation from immunoblots . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O B-SMALL_MOLECULE O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of insulin-induced Akt phosphorylation from immunoblots. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( E ) Real - time measurement ( F ) of insulin - induced fatty - acid uptake ( n = 4 ) . Representative Western blots are shown . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Real-time measurement and (F) quantification of insulin-induced fatty-acid uptake (n=4). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( C ) Acetylated α - tubulin in lines ( n = 3 ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Acetylated α-tubulin levels in cell lines (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( D ) Acetylated α - tubulin in isolated mature adipocytes from CTRL FAKO ( Mllt4 - / - ) mice ( n = 4 ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Acetylated α-tubulin levels in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( E ) IR abundance measured by immunoblotting following 4 hours pre - incubation with HDAC6 inhibitor ( Tubastatin A ) in differentiated adipocytes exposed to insulin ( 100nM ) stimulation for 15 min ( n = 4 ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) IR abundance measured by immunoblotting following 4 hours pre-incubation with the HDAC6 inhibitor (Tubastatin A) in differentiated adipocytes exposed to insulin (100nM) stimulation for 15 min (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( F ) Immunolocalization of IR ( green ) HDAC6 ( red ) in Afdn - KO Afdn - KO with re - expression FLAG - tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin . Nuclei were stained with DAPI ( blue ) . Co - localization was detected by appearance of pixels where red green overlap . Scale bars : 25 µm . Representative are shown . of IR co - localized with HDAC6 ( Mandler ' s co - localization coefficient ) was quantified ( 98 - 112 from two independent experiments ) . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-TISSUE I-TISSUE O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Immunolocalization of IR (green) and HDAC6 (red) in Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin. Nuclei were stained with DAPI (blue). Co-localization was detected by the appearance of white pixels where the red and green overlap. Scale bars: 25 µm. Representative images are shown. The ratio of IR co-localized with HDAC6 (Mandler's co-localization coefficient) was quantified (98-112 individual cells from two independent experiments). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( G ) Immunoprecipitation of IR followed by immunoblotting using an anti - acetyl - Lys antibody in adipocytes lacking Afadin or re - expressing Afadin , with or without insulin stimulation ( n = 1 ) . Representative Western blots are shown . Data are presented as means + ; Student ' s unpaired t - ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunoprecipitation of IR followed by immunoblotting using an anti-acetyl-Lys antibody in adipocytes lacking Afadin or re-expressing Afadin, with or without insulin stimulation (n=1). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( A ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (A) Body weight. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( B ) composition . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (B) Body composition. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( C ) Sirius Red of representative embedded BAT , sWAT vWAT from mice on HFD of adipocyte areas ( ~ 1000 adipocytes / genotype ) . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE O B-TISSUE I-TISSUE O B-ORGANISM O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (C) Sirius Red staining of representative embedded BAT, sWAT and vWAT from mice on HFD and quantification of adipocyte areas (~1000 adipocytes/genotype). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( D ) IPGTT after 12 of diet AUC . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (D) IPGTT after 12 weeks of diet and AUC. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( E ) ITT after 13 of diet AUC Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (E) ITT after 13 weeks of diet and AUC Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( F ) normalized to basal insulin . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O B-GENEPROD O O O O B-ORGANISM O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given high-fat diet (HFD) for 16 weeks (n=5-6). (F) normalized to basal insulin. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 ( n = 5 - 6 ) . ( G ) Plasma insulin 5 min after an IPGTT performed after 18 of diet . Data are presented as means + ; ANOVA with Tukey or Dunnet ' s post hoc : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O 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-TISSUE B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (G) Plasma insulin levels 5 min after an IPGTT performed after 18 weeks of diet. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( C ) Temporal of antigen - specific CD8 + T responses . Peripheral blood was obtained on days 4 , 7 , 14 , 21 , 42 88 post - immunisation with WT ( triangles ; n = 3 - 9 ) , CSPSIINFEKL ( orange squares ; n = 4 - 8 ) or UIS4SIINFEKL ( blue circles ; n = 4 - 10 ) sporozoites , or no ( diamonds ; n = 2 - 5 ) stained for Kb - SIINFEKL + CD11ahi CD8 + T . graph shows mean values ( ± ) from representative experiments . ( * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Welch ' s t - comparing CSPSIINFEKL UIS4SIINFEKL ) . See Appendix Table S1 for number of mice used per timepoint per group . See Appendix Table S4 for exact p - values .

O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

(C) Temporal analysis of antigen-specific CD8+ T cell responses. Peripheral blood was obtained on days 4, 7, 14, 21, 42 and 88 post-immunisation with WT (triangles; n=3-9), CSPSIINFEKL (orange squares; n=4-8) or UIS4SIINFEKL (blue circles; n=4-10) sporozoites, or no parasites (diamonds; n=2-5) and stained for Kb-SIINFEKL+ CD11ahi CD8+ T cells. Line graph shows mean values (±SEM) from representative experiments. (*, p<0.05; **, p<0.01; ***, p<0.001; Welch's t-test comparing CSPSIINFEKL and UIS4SIINFEKL). See Appendix Table S1 for the number of mice used per timepoint and per group. See Appendix Table S4 for exact p-values.


( D - H ) C57BL / 6 mice ( n = 4 per group ) , which received 2x106 CFSE - labelled OT - I splenocytes , were immunised with 10 , 000 γ - radiation attenuated WT , CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously . 5 days later , mice were sacrificed , spleens harvested splenocytes assessed for ( D ) CFSE dilution of CD8 + T , ( E ) CFSE dilution of antigen experienced Kb - SIINFEKL + CD11ahi CD8 + T stained ex vivo ( F - H ) for effector CD8 + T surface markers . Shown are flow cytometry plots of Kb - SIINFEKL co - with markers of effector : ( F ) CD11ahi , ( G ) CD62Llo ( H ) CD49dhi .

O O O O O O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O B-ORGANISM O O O B-TISSUE O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O

(D-H) C57BL/6 mice (n=4 per group), which received 2x106 CFSE-labelled OT-I splenocytes, were immunised with 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously. 5 days later, mice were sacrificed, spleens harvested and splenocytes assessed for (D) CFSE dilution of CD8+ T cells, (E) CFSE dilution of antigen experienced Kb-SIINFEKL+ CD11ahi CD8+ T cells and stained ex vivo (F-H) for effector CD8+ T cell surface markers. Shown are flow cytometry plots of Kb-SIINFEKL co-staining with markers of effector phenotypes: (F) CD11ahi, (G) CD62Llo and (H) CD49dhi.


( B Proportions numbers of ( B - Kb - SIINFEKL + CD8 + T were enumerated , or ( E - IFN - γ - secreting CD8 + T following restimulation ex vivo with SIINFEKL peptide were quantified . Flow cytometry plots show representative percentages of CD8 + T co - stained with CD11a ( B ) Kb - SIINFEKL or ( E ) IFN - γ .

O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O

(B Proportions and numbers of (B- Kb-SIINFEKL+ CD8+ T cells were enumerated, or (E- IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with CD11a and (B) Kb-SIINFEKL or (E) IFN-γ.


( B - C57BL / 6 mice received 10 , 000 γ - radiation attenuated WT , CSPSIINFEKL or UIS4SIINFEKL sporozoites , either intravenously ( n = 6 - 8 mice per group ) or ( E - intradermally ( n = 4 mice per group ) . Additional control mice did not receive sporozoites ( n = 6 - 8 intravenously , n = 4 intradermally ) . Spleens livers were harvested either at day 14 ( n = 5 livers n = 6 spleens for intravenous immunisation ) or day 42 ( n = 8 for intravenous immunisation ) , IFN - γ - secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified . Flow cytometry plots show representative percentages of CD8 + T co - stained with IFN - γ CD11a ( B , E ) .

O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(B- C57BL/6 mice received 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites, either intravenously (n=6-8 mice per group) or (E- intradermally (n=4 mice per group). Additional control mice did not receive sporozoites (n=6-8 intravenously, n=4 intradermally). Spleens and livers were harvested either at day 14 (n=5 livers and n=6 spleens for intravenous immunisation) or day 42 (n=8 for intravenous immunisation), and IFN-γ-secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with IFN-γ and CD11a (B, E).


( C ) upper panel of bar charts show of CD11ahi IFN - γ + CD8 + T lower panel absolute . Bar charts show mean values ( ± ) from representative experiments ( * * * , p < 0 . 001 ; one - way ANOVA with Tukey ' s multiple comparison ) . See Appendix Table S4 for exact p - values .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The upper panel of bar charts show the percentage of CD11ahi IFN-γ+ CD8+ T cells and the lower panel the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values.


( B , C ) Flow cytometry ( B ) scatter plots ( C ) represent CD8 + T derived from peripheral blood co - stained with IFN - γ CD11a , following ex vivo restimulation with SIINFEKL . Data information : Data are representative of two experiments performed with scatter plots showing mean values ( ± ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O 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, C) Flow cytometry (B) and scatter plots (C) represent CD8+ T cells derived from peripheral blood co-stained with IFN-γ and CD11a, following ex vivo restimulation with SIINFEKL. Data information: Data are representative of two experiments performed with scatter plots showing mean values (±SEM).


( D ) Protective efficacy as measured by quantitative real - time PCR . Groups of mice were vaccinated as described challenged 19 days later with 10 , 000 WT ( n = 7 non - vaccinated , n = 9 vaccinated ) , CSPSIINFEKL ( n = 6 non - vaccinated , n = 4 vaccinated ) or UIS4SIINFEKL sporozoites ( n = 3 non - vaccinated , n = 11 vaccinated ) . 42 hours later livers were removed , load was assessed by qPCR . Plots show relative load of mice in each condition ( * * , p < 0 . 01 ; Mann - Whitney U ) . See Appendix Table S4 for exact p - values . Data information : Data are representative of two experiments performed with scatter plots showing mean values ( ± ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O 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-GENEPROD O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Protective efficacy as measured by quantitative real-time PCR. Groups of mice were vaccinated as described and challenged 19 days later with 10,000 WT (n=7 non-vaccinated, n=9 vaccinated), CSPSIINFEKL (n=6 non-vaccinated, n=4 vaccinated) or UIS4SIINFEKL sporozoites (n=3 non-vaccinated, n=11 vaccinated). 42 hours later livers were removed, and parasite load was assessed by qPCR. Plots show the relative parasite load of mice in each condition (**, p<0.01; Mann-Whitney U test). See Appendix Table S4 for exact p-values. Data information: Data are representative of two experiments performed with scatter plots showing mean values (±SEM).


Representative of γH2AX immuno - FISH in troponin - C / α - actinin ( top left bottom left panels , respectively ) positive mouse CMs ( - troponin - C / α - actinin ; red - telo - FISH ; green - γH2AX ) . are Z - projections of 0 . 1μm stacks taken with a 100x oil objective . Scale bar : 20μm . Right panels represent a single Z - plane where co - localisation between a γH2AX foci telomere was observed , Scale bars 1μm . ( Below ) 3D reconstruction of Immuno - FISH using STED microscopy for γH2A . X telomeres in a CM from 30 month old mice . Scale bars as indicated scale same for both TAF examples using STED .

O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O

Representative images of γH2AX immuno-FISH in troponin-C/α-actinin (top left and bottom left panels, respectively) positive mouse CMs (white - troponin-C/α-actinin; red - telo-FISH; green - γH2AX). Images are Z-projections of 0.1μm stacks taken with a 100x oil objective. Scale bar: 20μm. Right panels represent a single Z-plane where co-localisation between a γH2AX foci and telomere was observed, Scale bars 1μm. (Below) 3D reconstruction of Immuno-FISH using STED microscopy for γH2A.X and telomeres in a CM from 30 month old mice. Scale bars as indicated and scale the same for both individual TAF examples using STED.


Mean number of TAF α - actinin positive CMs from C57BL / 6 mice . Data are mean ± of n = 4 - 7 mice per group .

O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-ORGANISM O O O

Mean number of TAF α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group.


α - actinin positive CMs from C57BL / 6 mice . Data are mean ± of n = 4 - 7 mice per group . 100 α - actinin positive CMs were quantified per mouse .

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α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.


Mean number of total γH2A . X foci in α - actinin positive CMs from C57BL / 6 mice . Data are mean ± of n = 4 - 7 mice per group . 100 α - actinin positive CMs were quantified per mouse .

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Mean number of total γH2A.X foci in α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.


% of γH2A . X foci - positive nuclei ( E ) in α - actinin positive CMs from C57BL / 6 mice . Data are mean ± of n = 4 - 7 mice per group . 100 α - actinin positive CMs were quantified per mouse .

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% of γH2A.X foci -positive nuclei (E) in α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.


Fold enrichment of γH2AX at telomere repeats by real - time PCR . Graph represents fold enrichment of γH2AX at telomeric repeats between IgG control , 3 30 month old whole mouse hearts . Data are mean ± of n = 3 mice per group .

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Fold enrichment of γH2AX at telomere repeats by real-time PCR. Graph represents fold enrichment of γH2AX at telomeric repeats between IgG control, 3 and 30 month old whole mouse hearts. Data are mean ± SEM of n=3 mice per age group.


Quantitative PCR - ELISA TRAP comparing telomerase activity of 3 30 month old C57BL / 6 mice whole heart lysates . Data are mean ± of n = 4 mice per group .

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Quantitative PCR-ELISA TRAP assay comparing telomerase activity of 3 and 30 month old C57BL/6 mice whole heart lysates. Data are mean ± SEM of n=4 mice per age group.


Histograms displaying distributions of telomere in CMs analysed by 3D Q - FISH in young ( 4 months ) old ( 30 months ) wild - type mice . Data are from n = 3 mice . > 100 CMs were analysed per mouse .

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Histograms displaying distributions of telomere intensity in CMs analysed by 3D Q-FISH in young (4 months) and old (30 months) wild-type mice. Data are from n=3 mice. > 100 CMs were analysed per mouse.


% of telomere FISH signal loss in 30 months old wild - type mice late generation Terc - / - mice ( 6 months old ) in comparison to 4 months old wild - type mice . Data are from n = 3 mice . > 100 CMs were analysed per mouse .

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% of telomere FISH signal loss in 30 months old wild-type mice and late generation Terc-/- mice (6 months old) in comparison to 4 months old wild-type mice. Data are from n=3 mice. >100 CMs were analysed per mouse.


Representative of mouse embryonic cardiomyocytes at days 0 , 3 , 5 10 days following 10Gy X - irradiation . Left panels represent troponin C - positive embryonic cardiomyocytes ( troponin C - magenta ; DAPI - blue ) . Middle panels display γH2AX foci ( green ) telomeres ( red ) in Z projections of 0 . 1µm slices , with arrows indicating co - localisation . Co - localising foci are amplified in right - panels ( amplified represent a single Z - plane where co - localisation was observed ) . Scale bars represent 10μm . Scale bars in single plane 500nm .

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Representative images of mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 days following 10Gy X-irradiation. Left panels represent troponin C-positive embryonic cardiomyocytes (troponin C - magenta; DAPI - light blue). Middle panels display γH2AX foci (green) and telomeres (red) in Z projections of 0.1µm slices, with white arrows indicating co-localisation. Co-localising foci are amplified in the right-hand panels (amplified images represent a single Z-plane where co-localisation was observed). Scale bars represent 10μm. Scale bars in single plane images 500nm.


( Left ) Mean number of both TAF non - TAF in troponin I - positive mouse embryonic cardiomyocytes at days 0 , 3 , 5 10 following 10Gy X - irradiation . Data are mean ± S . E . M of n = 3 independent experiments ; 30 - 50 troponin positive cardiomyocytes were analysed per experiment . ( Right ) Mean of γH2AX foci co - localising with telomeres ( % TAF ) in troponin C - positive mouse embryonic cardiomyocytes at days 0 , 3 , 5 10 following 10Gy X - irradiation . Statistical performed using one - way ANOVA ( Holm - Sidak method ) ; * P < 0 . 05 . Significant differences were found for mean number of non - TAF , but not for mean number of TAF .

O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O 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-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O B-SUBCELLULAR O

(Left) Mean number of both TAF and non-TAF in troponin I-positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10Gy X-irradiation. Data are mean ±S.E.M of n=3 independent experiments; 30-50 troponin positive cardiomyocytes were analysed per experiment. (Right) Mean percentage of γH2AX foci co-localising with telomeres (% TAF) in troponin C-positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10Gy X-irradiation. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); * P<0.05. Significant differences were found for mean number of non-TAF, but not for mean number of TAF.


Mean number of both TAF non - TAF in neonatal rat cardiomyocytes at days 0 , 3 , 5 , 10 days following treatment for 24h with H2O2 . Data are mean ± S . E . M of n = 3 . > 50 were quantified per condition .

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Mean number of both TAF and non-TAF in neonatal rat cardiomyocytes at days 0, 3, 5, 10 days following treatment for 24h with H2O2. Data are mean ±S.E.M of n=3. >50 cells were quantified per condition.