f8 ( a ) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide ( 48 - h cultures ) were immunoprecipitated with anti - ATG5 or antisurvivin antibodies . A physical interaction between ATG5 and survivin , absent in untreated or GFP - expressing control cells , was detected reciprocally . Full - length immunoblots are provided in Supplementary Fig . S12 . ( b ) Jurkat T cells overexpressing ATG5 - ΔNES exhibited no ATG5 / survivin molecular interaction . ( c ) The ATG5 - K130R mutant did show coprecipitation of survivin with ATG5 . ( d ) Calpain - mediated , truncated ATG5 ( tATG5 ) , that is , the N - terminal part of ATG5 , failed to bind survivin . The ATG5 - K130R mutant was the control . ( e ) Confocal microscopy . MDA - MA - 231 cells were etoposide treated or transduced as indicated ( 48 - h cultures ) and stained . In untreated cells , survivin and ATG5 were in the cytosol with no colocalization . ATG5 - transduced cells exhibited ATG5 / survivin colocalization in the nucleus . ATG5 - ΔNES - transduced cells showed survivin , but not ATG5 , in the nuclei of cycling cells . Numerical analysis was performed and Pearson ' s correlation coefficients are indicated in the images . Scale bar , 10 μm . Right : Statistical analysis ( analysis of variance ) is presented in which the results of ten representative cells within each group were integrated . Values are means ± s . d . ( f ) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5 . Neither ATG5 nor HSP90 were detectable in the Aurora B - containing immune precipitate , but ATG5 molecularly interacted with survivin . Results representative of three independent experiments . ( g ) Confocal microscopy . HeLa cells treated with etoposide or transduced as indicated ( 48 - h cultures ) were stained with centromere - specific antiserum and anti - Aurora B ( top panels ) or antisurvivin antibodies ( lower panels ) . In untreated or ATG5 - ΔNES transduced cells , both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres . In contrast , etoposide - treated or ATG5 - transduced cells recruited much less Aurora B to prometaphase centromeres . Such recruitment , however , was observed during anaphase . Compared with cells in normal mitosis , survivin , although reduced , was recruited to centromeres in prometaphase , but , similar to Aurora B , remained attached to centromeres during anaphase . Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects . Colocalization images were prepared using Imaris software . Numerical analysis was performed and correlation coefficients are indicated . Scale bar , 2 μm . ( h ) Confocal microscopy . Sections of lung ( non - small - cell lung cancer ) and oesophageal carcinoma stained for ATG5 and survivin expression . In untreated patients , ATG5 expression in cancer cells was slight and mainly cytosolic . Following radio - and / or chemotherapy , ATG5 was preferentially expressed in the nucleus and colocalized with survivin . Overlay images present the average Pearson ' s correlation coefficient as calculated with Imaris . Results are representative of three independent experiments . Scale bar , 10 μm .

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

f8(a) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide (48-h cultures) were immunoprecipitated with anti-ATG5 or antisurvivin antibodies. A physical interaction between ATG5 and survivin, absent in untreated or GFP-expressing control cells, was detected reciprocally. Full-length immunoblots are provided in Supplementary Fig. S12.(b) Jurkat T cells overexpressing ATG5-ΔNES exhibited no ATG5/survivin molecular interaction. (c) The ATG5-K130R mutant did show coprecipitation of survivin with ATG5.(d) Calpain-mediated, truncated ATG5 (tATG5), that is, the N-terminal part of ATG5, failed to bind survivin. The ATG5-K130R mutant was the control.(e) Confocal microscopy. MDA-MA-231 cells were etoposide treated or transduced as indicated (48-h cultures) and stained. In untreated cells, survivin and ATG5 were in the cytosol with no colocalization. ATG5-transduced cells exhibited ATG5/survivin colocalization in the nucleus. ATG5-ΔNES-transduced cells showed survivin, but not ATG5, in the nuclei of cycling cells. Numerical analysis was performed and Pearson's correlation coefficients are indicated in the images. Scale bar, 10 μm. Right: Statistical analysis (analysis of variance) is presented in which the results of ten representative cells within each group were integrated. Values are means±s.d.(f) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5. Neither ATG5 nor HSP90 were detectable in the Aurora B-containing immune precipitate, but ATG5 molecularly interacted with survivin. Results representative of three independent experiments.(g) Confocal microscopy. HeLa cells treated with etoposide or transduced as indicated (48-h cultures) were stained with centromere-specific antiserum and anti-Aurora B (top panels) or antisurvivin antibodies (lower panels). In untreated or ATG5-ΔNES transduced cells, both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres. In contrast, etoposide-treated or ATG5-transduced cells recruited much less Aurora B to prometaphase centromeres. Such recruitment, however, was observed during anaphase. Compared with cells in normal mitosis, survivin, although reduced, was recruited to centromeres in prometaphase, but, similar to Aurora B, remained attached to centromeres during anaphase. Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects. Colocalization images were prepared using Imaris software. Numerical analysis was performed and correlation coefficients are indicated. Scale bar, 2 μm.(h) Confocal microscopy. Sections of lung (non-small-cell lung cancer) and oesophageal carcinoma stained for ATG5 and survivin expression. In untreated patients, ATG5 expression in cancer cells was slight and mainly cytosolic. Following radio- and/or chemotherapy, ATG5 was preferentially expressed in the nucleus and colocalized with survivin. Overlay images present the average Pearson's correlation coefficient as calculated with Imaris. Results are representative of three independent experiments. Scale bar, 10 μm.


Figure 1A - H Effect of CCCP on the localisation of Noc and NocNΔ10 . Cellular localisation of Noc ‐ mYFP ( DWA206 ) and NocNΔ10 ‐ mYFP ( DWA382 ) either with no additions ( NA ) or after CCCP treatment ( 5 min ; 100 μM ) , as indicated . Scale bar , 2 . 5 μm . I - L Effect of Noc overproduction on cell division . Exponentially growing cultures of DWA119 ( Δnoc , Pspac ( hy ) ‐ noc ) and DWA282 ( Δnoc , Pspac ( hy ) ‐ nocNΔ10 ) were examined after growth for 1 h with 1 mM IPTG . Cell membranes and DNA were stained with FM5 ‐ 95 and DAPI , respectively . Insets show corresponding phase contrast light microscopy images . Scale bar , 5 μm . M Complementation of noc in a Δnoc ΔminCD background . Strains DWA564 ( Δnoc , ΔminCD , Pxyl ‐ noc ‐ myfp ) and 566 ( Δnoc , ΔminCD , Pxyl ‐ nocNΔ10 ‐ myfp ) were streaked on nutrient agar ( NA ) plates in the presence of 0 . 5 % w / v xylose and incubated at 30 and 39 ° C , as indicated . N Effect of Noc and NocNΔ10 overproduction on sporulation . Strains DWA119 ( Δnoc , Pspac ( hy ) ‐ noc ) and DWA282 ( Δnoc , Pspac ( hy ) ‐ nocNΔ10 ) were streaked on NA plates in the absence and presence of 1 mM IPTG , as indicated , and photographed after 48 h at 37 ° C . O Western blot analysis of cellular fractions ( T , total ; C , cytosolic ; M , membrane ) of strains DWA119 and 282 expressing Noc or NocNΔ10 , respectively . Proteins were detected using polyclonal antibodies against Noc , DnaA and PBP2B . Antibodies were used at a dilution of 1 : 10 , 000 .

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

Figure 1A-H Effect of CCCP on the localisation of Noc and NocNΔ10. Cellular localisation of Noc‐mYFP (DWA206) and NocNΔ10‐mYFP (DWA382) either with no additions (NA) or after CCCP treatment (5 min; 100 μM), as indicated. Scale bar, 2.5 μm.I-L Effect of Noc overproduction on cell division. Exponentially growing cultures of DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were examined after growth for 1 h with 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Insets show corresponding phase contrast light microscopy images. Scale bar, 5 μm.M Complementation of noc in a Δnoc ΔminCD background. Strains DWA564 (Δnoc, ΔminCD, Pxyl‐noc‐myfp) and 566 (Δnoc, ΔminCD, Pxyl‐nocNΔ10‐myfp) were streaked on nutrient agar (NA) plates in the presence of 0.5% w/v xylose and incubated at 30 and 39°C, as indicated.N Effect of Noc and NocNΔ10 overproduction on sporulation. Strains DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were streaked on NA plates in the absence and presence of 1 mM IPTG, as indicated, and photographed after 48 h at 37°C.O Western blot analysis of cellular fractions (T, total; C, cytosolic; M, membrane) of strains DWA119 and 282 expressing Noc or NocNΔ10, respectively. Proteins were detected using polyclonal antibodies against Noc, DnaA and PBP2B. Antibodies were used at a dilution of 1:10,000.


Figure 2C - L Effects of N ‐ terminal substitutions on Noc localisation , in strains : DWA211 ( F5E ) , 318 ( F9E ) , 316 ( K2E ) , 212 ( R7E ) , 322 ( F5A ) , 323 ( F8A ) , 325 ( F9A ) , 206 ( WT ) , 328 ( S4A ) and 329 ( S4L ) , as indicated . Insets show the corresponding phase contrast light microscopy images . Scale bar , 5 μm .

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

Figure 2C-L Effects of N‐terminal substitutions on Noc localisation, in strains: DWA211 (F5E), 318 (F9E), 316 (K2E), 212 (R7E), 322 (F5A), 323 (F8A), 325 (F9A), 206 (WT), 328 (S4A) and 329 (S4L), as indicated. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.


Figure 3B Growth of strains DWA350 ( Δnoc ΔminCD ) and DWA307 ( Δnoc ΔminCD , Pxyl ‐ HCVAH ‐ NocNΔ10 ) on nutrient agar plates at 30 and 48 ° C in the absence and presence of 0 . 5 % w / v xylose , as indicated . C - F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30 ° C ( C and D ) and at 42 ° C in LB + 0 . 5 % w / v xylose ( E and F ) . Arrowheads indicate minicells . Cell membranes were stained with FM5 - 95 . Insets show the corresponding phase contrast light microscopy images . Scale bar , 5 μm . G , H Cellular localisation of HCVAH ‐ NocNΔ10 ‐ YFP ( G ) in strain DWA193 ( Δnoc , Pxyl ‐ HCVAH ‐ nocNΔ10 ‐ yfp ) and overlay showing DAPI ‐ stained DNA ( H ) . The strain was grown at 30 ° C in CH medium . Inset shows the corresponding phase contrast light microscopy image . Scale bar , 5 μm .

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

Figure 3B Growth of strains DWA350 (Δnoc ΔminCD) and DWA307 (Δnoc ΔminCD, Pxyl‐HCVAH‐NocNΔ10) on nutrient agar plates at 30 and 48°C in the absence and presence of 0.5% w/v xylose, as indicated.C-F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30°C (C and D) and at 42°C in LB + 0.5% w/v xylose (E and F). Arrowheads indicate minicells. Cell membranes were stained with FM5-95. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.G, H Cellular localisation of HCVAH‐NocNΔ10‐YFP (G) in strain DWA193 (Δnoc, Pxyl‐HCVAH‐nocNΔ10‐yfp) and overlay showing DAPI‐stained DNA (H). The strain was grown at 30°C in CH medium. Inset shows the corresponding phase contrast light microscopy image. Scale bar, 5 μm.


Figure 4A - F Localisation of ParB ‐ box mutants . Cellular localisation of ( A ) Noc ‐ mYFP ( DWA206 ) , ( B ) NocQ68R ‐ mYFP ( DWA285 ) , ( C ) NocG86S ‐ mYFP ( DWA286 ) , ( D ) NocR88A ‐ mYFP ( DWA545 ) , ( E ) NocR89A ‐ mYFP ( DWA546 ) and ( F ) NocR91A ‐ mYFP ( DWA547 ) . Insets show the corresponding phase contrast images . Scale bar , 5 μm . G Ability of ParB ‐ box mutants to rescue the growth defect of Δnoc ΔminCD . Strains DWA564 ( Pxyl ‐ noc ‐ myfp ) , 590 ( Pxyl ‐ nocQ68R ‐ myfp ) , 568 ( Pxyl ‐ nocG86S ‐ myfp ) , 598 ( Pxyl ‐ nocR88A ‐ myfp ) , 600 ( Pxyl ‐ nocR89A ‐ myfp ) and 602 ( Pxyl ‐ nocR91A ‐ myfp ) were streaked on plates containing 0 . 5 % w / v xylose and incubated for 18 h at either 30 or 39 ° C , as indicated , before being photographed . H - O ParB ‐ box mutants are dominant ‐ negative . Cells of strains DWA362 ( ΔminCD , Pspac ( hy ) ‐ noc ) , 363 ( ΔminCD , Pspac ( hy ) ‐ nocNΔ10 ) , 364 ( ΔminCD , Pspac ( hy ) ‐ nocQ68R ) and 365 ( ΔminCD , Pspac ( hy ) ‐ nocG86S ) were examined after growth for 2 h at 42 ° C with either no additions ( NA ) ( H - K ) or in the presence of 1 mM IPTG ( L - O ) , as indicated . Cell membranes were stained with FM5 ‐ 95 . Insets show the corresponding phase contrast images . Scale bar , 5 μm .

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

Figure 4A-F Localisation of ParB‐box mutants. Cellular localisation of (A) Noc‐mYFP (DWA206), (B) NocQ68R‐mYFP (DWA285), (C) NocG86S‐mYFP (DWA286), (D) NocR88A‐mYFP (DWA545), (E) NocR89A‐mYFP (DWA546) and (F) NocR91A‐mYFP (DWA547). Insets show the corresponding phase contrast images. Scale bar, 5 μm.G Ability of ParB‐box mutants to rescue the growth defect of Δnoc ΔminCD. Strains DWA564 (Pxyl‐noc‐myfp), 590 (Pxyl‐nocQ68R‐myfp), 568 (Pxyl‐nocG86S‐myfp), 598 (Pxyl‐nocR88A‐myfp), 600 (Pxyl‐nocR89A‐myfp) and 602 (Pxyl‐nocR91A‐myfp) were streaked on plates containing 0.5% w/v xylose and incubated for 18 h at either 30 or 39°C, as indicated, before being photographed.H-O ParB‐box mutants are dominant‐negative. Cells of strains DWA362 (ΔminCD, Pspac(hy)‐noc), 363 (ΔminCD, Pspac(hy)‐nocNΔ10), 364 (ΔminCD, Pspac(hy)‐nocQ68R) and 365 (ΔminCD, Pspac(hy)‐nocG86S) were examined after growth for 2 h at 42°C with either no additions (NA) (H-K) or in the presence of 1 mM IPTG (L-O), as indicated. Cell membranes were stained with FM5‐95. Insets show the corresponding phase contrast images. Scale bar, 5 μm.


Figure 5A - C Representative images of TetR ‐ mCherry in cells lacking ( A ) or containing ( B and C ) the NBS plasmid pDWA117 . Strains DWA427 ( A ) and 429 ( B and C ) ( both strains contain Pspac ‐ noc ) were examined after growth for 2 h in the absence ( A and B ) and presence ( C ) of 1 mM IPTG . DNA was stained with DAPI . D - G Representative images showing the localisation of ( D ) Noc ‐ mYFP ( DWA519 ) , ( E ) NocNΔ10 ‐ mYFP ( DWA522 ) , ( F ) NocQ68R ‐ mYFP ( DWA520 ) and ( G ) NocG86S ‐ mYFP ( DWA521 ) , in the presence of the NBS plasmid pSG4929 .

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

Figure 5A-C Representative images of TetR‐mCherry in cells lacking (A) or containing (B and C) the NBS plasmid pDWA117. Strains DWA427 (A) and 429 (B and C) (both strains contain Pspac‐noc) were examined after growth for 2 h in the absence (A and B) and presence (C) of 1 mM IPTG. DNA was stained with DAPI.D-G Representative images showing the localisation of (D) Noc‐mYFP (DWA519), (E) NocNΔ10‐mYFP (DWA522), (F) NocQ68R‐mYFP (DWA520) and (G) NocG86S‐mYFP (DWA521), in the presence of the NBS plasmid pSG4929.


Figure 6A , B Effects of Noc overproduction on cell division and nucleoid morphology in E . coli . Cells of strain DWA261 carrying pDWA37 ( PA1 / 04 / 03 ‐ noc ) were examined after growth in LB with either no additions ( A ) or after induction for 1 h with 1 mM IPTG ( B ) . C - E Effects of overproduction of Noc variants on nucleoid morphology . Cells of strains DWA266 ( PA1 / 04 / 03 ‐ nocNΔ10 ) ( C ) , 270 ( PA1 / 04 / 03 ‐ HCVAH ‐ nocNΔ10 ) ( D ) and 267 ( PA1 / 04 / 03 ‐ nocCΔ50 ) ( E ) carrying plasmids for the overproduction of the indicated mutants ( see cartoons underneath panels ) were grown in LB and examined after growth for 1 h in the presence of 1 mM IPTG . Cell membranes and DNA were stained with FM5 ‐ 95 and DAPI , respectively . Scale bar , 5 μm .

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

Figure 6A, B Effects of Noc overproduction on cell division and nucleoid morphology in E. coli. Cells of strain DWA261 carrying pDWA37 (PA1/04/03‐noc) were examined after growth in LB with either no additions (A) or after induction for 1 h with 1 mM IPTG (B).C-E Effects of overproduction of Noc variants on nucleoid morphology. Cells of strains DWA266 (PA1/04/03‐nocNΔ10) (C), 270 (PA1/04/03‐HCVAH‐nocNΔ10) (D) and 267 (PA1/04/03‐nocCΔ50) (E) carrying plasmids for the overproduction of the indicated mutants (see cartoons underneath panels) were grown in LB and examined after growth for 1 h in the presence of 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Scale bar, 5 μm.


Figure 7A , B Effects of Spo0J and Noc30 ‐ Spo0J overproduction on cell division and nucleoid morphology in E . coli . Cells of strains DWA271 ( PA1 / 04 / 03 ‐ spo0J ) ( A ) and 272 ( PA1 / 04 / 03 ‐ noc ‐ spo0J ) ( B ) were grown in LB in the presence of 1 mM IPTG , to induce the expression of either Spo0J ( A ) or the Noc ‐ Spo0J hybrid ( B ) , and were examined 1 h post ‐ induction . Cell membranes and DNA were stained with FM5 - 95 and DAPI , respectively . Scale bar , 5 μm .

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

Figure 7A, B Effects of Spo0J and Noc30‐Spo0J overproduction on cell division and nucleoid morphology in E. coli. Cells of strains DWA271 (PA1/04/03‐spo0J) (A) and 272 (PA1/04/03‐noc‐spo0J) (B) were grown in LB in the presence of 1 mM IPTG, to induce the expression of either Spo0J (A) or the Noc‐Spo0J hybrid (B), and were examined 1 h post‐induction. Cell membranes and DNA were stained with FM5-95 and DAPI, respectively. Scale bar, 5 μm.


Figure 2 ( A ) Volcano plot shows genes where RNA reads are statistically significant under - or over - expressed in 5 RIRCD affected individuals versus 6 controls resulted from DESeq2 analysis . Decreased genes are represented in red , the up - regulated ones are in green while blue dots highlight most outlying genes ( defined by absolute ( log2 ( fold change ) + log10 ( padj ) ) > 10 ) . Genes that were not statistically significant are represented in black . Grey and black gene names indicate top 30 % of the most dysregulated genes with a highlight on ones involved in metabolism . ( D ) Relevant functional changes in gene expression showing the major genes that return to baseline or contribute to the recovery in RIRCD patients . The data depicted is between the affected and recovered biopsies of the same patient ( F6 / 1M ) . ( E ) Comparative proteome profiling of proteins that are significantly under - or over - expressed in RIRCD affected versus control individuals ( p - Anova < 0 . 05 - statistically significant - horizontal line ) . Proteins which are decreased are represented in red while the up - regulated ones are in green while proteins that did not reach statistical significance threshold are marked in black . Vertical lines are delimiting the regulated proteins resulted from the proteomics analysis ( the cut - off values were determined based on the 2 × standard deviation and the normal distribution from all identified protein ' s log2 ratio in which the bell curve is symmetric around the mean , therefore , an average log2 ratio of a protein which < − 2 . 2 or > 0 . 98 was considered as regulated ) .

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

Figure 2(A) Volcano plot shows genes where RNA reads are statistically significant under- or over-expressed in 5 RIRCD affected individuals versus 6 controls resulted from DESeq2 analysis. Decreased genes are represented in red, the up-regulated ones are in green while blue dots highlight most outlying genes (defined by absolute (log2(fold change) + log10(padj))>10).Genes that were not statistically significant are represented in black. Grey and black gene names indicate top 30% of the most dysregulated genes with a highlight on ones involved in metabolism.(D) Relevant functional changes in gene expression showing the major genes that return to baseline or contribute to the recovery in RIRCD patients. The data depicted is between the affected and recovered biopsies of the same patient (F6/1M).(E) Comparative proteome profiling of proteins that are significantly under- or over-expressed in RIRCD affected versus control individuals (p-Anova <0.05-statistically significant - horizontal line). Proteins which are decreased are represented in red while the up-regulated ones are in green while proteins that did not reach statistical significance threshold are marked in black. Vertical lines are delimiting the regulated proteins resulted from the proteomics analysis (the cut-off values were determined based on the 2× standard deviation and the normal distribution from all identified protein's log2 ratio in which the bell curve is symmetric around the mean, therefore, an average log2 ratio of a protein which <−2.2 or > 0.98 was considered as regulated).


Figure 3 ( A ) Western blotting analysis depicts mitochondrial proteins ( NDUFA9 - Complex I , SDHA - Complex II , MTCO1 - Complex IV and MTCO2 - Complex IV ) levels in control derived muscle ( n = 3 ) , patient derived muscle during the symptomatic phase ( n = 3 ) and patient derived muscle after recovery ( n = 1 ) . VCL was used as a loading control while the densitometry analysis was done based on the mitochondrial protein TOM20 . ( B ) Densitometry analysis of the immunoblotting from ( A ) showing an increase in SDHA ( Complex II ) , while NDUFA9 ( Complex I ) probably as a compensatory mechanism and MT - CO2 ( Complex IV ) show significant reductions as the majority of mitochondrial encoded proteins belong to this two complexes , thus being destabilized by the defective mitochondrial mistranslation . ( C ) Mitochondrial DNA copy numbers were determined in affected ( n = 3 ) and recovered individuals ( n = 2 ) and compared to control individuals ( n = 4 , 3 months , 3 years , 4 years and 7 years ) using qPCR showing a significant increase in recovered muscle . ( D ) Western blotting analysis of proteins involved in the amino - acid sensing and integrated stress response : GCN2 , SLC7A11 and ATF4 in control derived muscle ( n = 3 ) , patient derived muscle during the symptomatic phase ( n = 3 ) and patient derived muscle after recovery ( n = 1 ) . All samples were normalized to VCL which was used as a loading control . ( E ) Densitometry analysis of proteins from ( D ) showing a significant increase of major transducers of ISR such as GCN2 and ATF4 . Consistent with ISR activation is also the increase in SLC7A1 . ( F ) Transcriptomic pathways related to ATFs and ISR changed in affected patients compared to controls ( RIRCD n = 5 ; controls n = 6 ) and in recovered muscle compared to affected muscle ( n = 1 , affected and recovered muscle was collected from the same individual and was analysed using 4 technical replicates ) and schematic representation of the metabolic pathways impacted in patient muscle ( bottom , right panel ) .

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

Figure 3(A) Western blotting analysis depicts mitochondrial proteins (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) levels in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). VCL was used as a loading control while the densitometry analysis was done based on the mitochondrial protein TOM20. (B) Densitometry analysis of the immunoblotting from (A) showing an increase in SDHA (Complex II), while NDUFA9 (Complex I) probably as a compensatory mechanism and MT-CO2 (Complex IV) show significant reductions as the majority of mitochondrial encoded proteins belong to this two complexes, thus being destabilized by the defective mitochondrial mistranslation.(C) Mitochondrial DNA copy numbers were determined in affected (n=3) and recovered individuals (n=2) and compared to control individuals (n=4, 3 months, 3 years, 4 years and 7 years) using qPCR showing a significant increase in recovered muscle.(D) Western blotting analysis of proteins involved in the amino-acid sensing and integrated stress response: GCN2, SLC7A11 and ATF4 in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). All samples were normalized to VCL which was used as a loading control. (E) Densitometry analysis of proteins from (D) showing a significant increase of major transducers of ISR such as GCN2 and ATF4. Consistent with ISR activation is also the increase in SLC7A1.(F) Transcriptomic pathways related to ATFs and ISR changed in affected patients compared to controls (RIRCD n=5; controls n=6) and in recovered muscle compared to affected muscle (n=1, affected and recovered muscle was collected from the same individual and was analysed using 4 technical replicates) and schematic representation of the metabolic pathways impacted in patient muscle (bottom, right panel).


Figure 4 ( A ) Patient n = 4 ( F7 / 7F - healthy carrier , F7 / 2M - RIRCD , F7 / 5F - healthy carrier , F7 / 1M - RIRCD without a clear second mutation ) and control fibroblasts ( n = 2 ) were grown for 12 days in media containing 0 . 02 mM cysteine and 5 % dialyzed FBS . Proteins belonging to mitochondrial complexes ( NDUFA9 - Complex I , SDHA - Complex II , MTCO1 - Complex IV and MTCO2 - Complex IV ) were analysed via Western Blotting . VCL was used as a loading control and the densitometry analysis was done based on the mitochondrial proteinTOM20 . ( B ) Densitometry analysis of proteins from ( A ) showing no significant changes in OXPHOS proteins upon glutamine / glutamic acid depletion . Control bar represents an average of the two control fibroblast lines employed ( F008 and F154 ) . ( C ) Patient ( F7 / 7F - healthy carrier , F7 / 2M - RIRCD , F7 / 5F - healthy carrier , F7 / 1M - RIRCD without a clear second mutation ) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5 % dialyzed FBS . Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting . VCL was used as a loading control while the densitometry analysis ( D ) was done based on the mitochondrial proteinTOM20 . Control bar represents an average of the two control fibroblast lines employed ( F008 and F154 ) . Although we could observe significant changes upon cysteine depletion in all conditions , the phenotype was exacerbated in the digenic fibroblasts compared to the controls .

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

Figure 4(A) Patient n= 4 (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts (n=2) were grown for 12 days in media containing 0.02 mM cysteine and 5% dialyzed FBS. Proteins belonging to mitochondrial complexes (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) were analysed via Western Blotting. VCL was used as a loading control and the densitometry analysis was done based on the mitochondrial proteinTOM20. (B) Densitometry analysis of proteins from (A) showing no significant changes in OXPHOS proteins upon glutamine/ glutamic acid depletion. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). (C) Patient (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5% dialyzed FBS. Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting. VCL was used as a loading control while the densitometry analysis (D) was done based on the mitochondrial proteinTOM20. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). Although we could observe significant changes upon cysteine depletion in all conditions, the phenotype was exacerbated in the digenic fibroblasts compared to the controls.


Figure 5 ( A ) Volcano plot analysis of transcriptomic changes where statistically significant genese are delimited by a horizontal line . Decreased genes are represented in red while the up - regulated ones are in green , while genes that were not statistically significant are represented in black . ( B ) Analysis of altered mitochondrial gene expression resulted from our RNAseq showing that affected genes and their coding proteins carry a high numbers of glutamic acid and glutamine residues , in accordance to the mitochondrial translational defect .

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

Figure 5(A) Volcano plot analysis of transcriptomic changes where statistically significant genese are delimited by a horizontal line. Decreased genes are represented in red while the up-regulated ones are in green, while genes that were not statistically significant are represented in black.(B) Analysis of altered mitochondrial gene expression resulted from our RNAseq showing that affected genes and their coding proteins carry a high numbers of glutamic acid and glutamine residues, in accordance to the mitochondrial translational defect.


Figure 1FP titrations show binding of WT α - actinin - 1 _ EF34 to IQ peptides WT ( black ) and K1647E ( red ) , of α - actinin - 1 _ EF34 mutant E847K / E851K to IQ peptides WT ( green ) and K1647E ( purple ) , of full - length α - actinin - 1 to IQ WT ( o ) , and lack of IQ binding to α - actinin - 1 _ CH1 - CH2 ( + ) and α - actinin - 1 _ EF12 ( x ; see Table 2 for binding parameters and standard errors ) . FP titrations showing binding of wild type α - actinin - 1 _ EF34 to IQ peptides WT ( black ) and I1654A ( purple ) and of ACTN1 _ EF34 mutant F833 to IQ WT ( red ; see Table 2 for binding parameters and standard errors ) .

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

Figure 1FP titrations show binding of WT α-actinin-1_EF34 to IQ peptides WT (black) and K1647E (red), of α-actinin-1_EF34 mutant E847K/E851K to IQ peptides WT (green) and K1647E (purple), of full-length α-actinin-1 to IQ WT (o), and lack of IQ binding to α-actinin-1_CH1-CH2 (+) and α-actinin-1_EF12 (x; see Table 2 for binding parameters and standard errors). FP titrations showing binding of wild type α-actinin-1_EF34 to IQ peptides WT (black) and I1654A (purple) and of ACTN1_EF34 mutant F833 to IQ WT (red; see Table 2 for binding parameters and standard errors).


Figure 2FP titrations showing binding of apoCaM to IQ peptides WT ( black ) and K1662E ( red ) and of apoCaM mutant E88K to IQ WT ( green ) and IQ peptide K1662E ( purple , " charge inversion " ; see Table 4 for binding parameters and standard errors ) . FP titrations showing binding of apo - CaM to IQ peptides WT ( black ) , I1654A ( red ) , and F1658A ( green ) and of apoCaM mutant F90A to IQ WT ( blue ; see Table 4 for binding parameters and standard errors ) .

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

Figure 2FP titrations showing binding of apoCaM to IQ peptides WT (black) and K1662E (red) and of apoCaM mutant E88K to IQ WT (green) and IQ peptide K1662E (purple, "charge inversion"; see Table 4 for binding parameters and standard errors). FP titrations showing binding of apo-CaM to IQ peptides WT (black), I1654A (red), and F1658A (green) and of apoCaM mutant F90A to IQ WT (blue; see Table 4 for binding parameters and standard errors).


Figure 3HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A and cultured for 22 - 24h before cell attached patch recording in 110 mM Ba2 + . Single CaV1 . 2 channel recordings of CaV1 . 2 WT and K1647A . Holding potential ( HP ) was - 80 mV and test potential ( TP ) 0 mV . Shown are 10 consecutive sweeps from representative experiments ( see Fig . EV 4 for more sweeps ) . Dot plots and means + SEM for availability ( i . e . , likelihood that a sweep had at least one event ; c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( e ) , and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment ( g ) ( * * p < 0 . 01 , * * * p < 0 . 001 compared to WT ; one - way ANOVA with Bonferroni post - hoc test ( c ) or Welch ANOVA with Tamhane T2 test ( d - g ) ; n = 7 to 35 see Table 5 for more details ) .

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

Figure 3HEK293 cells were transfected with α11.2, α2δ-1, and β2A and cultured for 22-24h before cell attached patch recording in 110 mM Ba2+. Single CaV1.2 channel recordings of CaV1.2 WT and K1647A. Holding potential (HP) was -80 mV and test potential (TP) 0 mV. Shown are 10 consecutive sweeps from representative experiments (see Fig. EV 4 for more sweeps). Dot plots and means + SEM for availability (i.e., likelihood that a sweep had at least one event; c), NPo (d), Po (e), unitary current amplitude i (e), and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment (g) (**p<0.01, ***p<0.001 compared to WT; one-way ANOVA with Bonferroni post-hoc test (c) or Welch ANOVA with Tamhane T2 test (d-g); n = 7 to 35 see Table 5 for more details).


Figure 4HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A plus , if indicated , α - actinin - 1 before cell attached patch recording . Representative single CaV1 . 2 channel recordings of WT CaV1 . 2 alone or with WT or E847K / E851K mutant α - actinin - 1 . Mean assemble averages for all experiments for each combination . Dot plots and means + SEM for availability ( c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( f ) , and the mean of the current of a single channel calculated from the ensemble averages of each experiment ( g ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 001 ; unpaired t - test ; n = 13 to 35 ; see Table 6 for more details ) .

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

Figure 4HEK293 cells were transfected with α11.2, α2δ-1, and β2A plus, if indicated, α-actinin-1 before cell attached patch recording. Representative single CaV1.2 channel recordings of WT CaV1.2 alone or with WT or E847K/E851K mutant α-actinin-1. Mean assemble averages for all experiments for each combination. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and the mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (*p<0.05, **p<0.01, ****p<0.001; unpaired t-test; n = 13 to 35; see Table 6 for more details).


Figure 5HEK293 cells were transfected with α11 . 2 with the K1647E mutation , α2δ - 1 , and β2A plus α - actinin - 1 WT or E847K / E851K before cell attached patch recording . Representative single CaV1 . 2 channel recordings of CaV1 . 2 K1647E with WT or E847K / E851K α - actinin - 1 . Mean assemble averages for all experiments for both combinations . Dot plots and means + SEM for availability ( c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( f ) , and mean of the current of a single channel calculated from the ensemble averages of each experiment ( g ) ( * * p < 0 . 01 ; unpaired t - test ; n = 16 to 30 ; see Suppl . Fig . 8 and Table 6 for more details ) .

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

Figure 5HEK293 cells were transfected with α11.2 with the K1647E mutation, α2δ-1, and β2A plus α-actinin-1 WT or E847K/E851K before cell attached patch recording. Representative single CaV1.2 channel recordings of CaV1.2 K1647E with WT or E847K/E851K α-actinin-1. Mean assemble averages for all experiments for both combinations. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (**p<0.01; unpaired t-test; n = 16 to 30; see Suppl. Fig. 8 and Table 6 for more details).


Figure 6HEK293 cells were transfected with WT α11 . 2 , α2δ - 1 , and β2A + WT α - actinin - 1 and cultured for 22 - 24h before surface biotinylation . Shown are representative immunoblots of NeutrAvidin pull - down samples ( from lysate containing 600 μg protein ) and total lysate samples containing 20 μg protein . α11 . 2 was detected with an antibody against its HA tag ( top ) , which is present in all constructs used throughout this work . Pull - down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull - down samples . Immunoblotting for vinculin ( middle ) and tubulin ( bottom ) indicated that comparable amounts of these intracellular control proteins were present in lysate samples . Their absence in pull - down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation . Bar graph shows means + SEM of the pull - down immunosignals in mutants relative to surface labelling of control α11 . 2 samples lacking α - actinin - 1 co - expression ( mean set to 100 % ; see Methods ; * * p < 0 . 01 two - tailed t - test ; n = 7 ) . HEK293 cells were transfected with WT or K1647E mutant α11 . 2 , α2δ - 1 , and β2A , and WT or E847K / E851K mutant α - actinin - 1 , or CFP alone as negative control and cultured for 22 - 24h before surface biotinylation . Shown are representative immunoblots of pull - down and lysate samples as in ( a ) . Bar graph shows means + SEM of the pull - down immunosignals in mutants relative to surface labelling in the WT / WT control ( mean set to 100 % ; see Methods ) . * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with Tukey post hoc test ; n = 4 - 5 ) .

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

Figure 6HEK293 cells were transfected with WT α11.2, α2δ-1, and β2A + WT α-actinin-1 and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of NeutrAvidin pull-down samples (from lysate containing 600 μg protein) and total lysate samples containing 20 μg protein. α11.2 was detected with an antibody against its HA tag (top), which is present in all constructs used throughout this work. Pull-down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull-down samples. Immunoblotting for vinculin (middle) and tubulin (bottom) indicated that comparable amounts of these intracellular control proteins were present in lysate samples. Their absence in pull-down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation. Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling of control α11.2 samples lacking α-actinin-1 co-expression (mean set to 100%; see Methods; **p<0.01 two-tailed t-test; n = 7).HEK293 cells were transfected with WT or K1647E mutant α11.2, α2δ-1, and β2A, and WT or E847K/E851K mutant α-actinin-1, or CFP alone as negative control and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of pull-down and lysate samples as in (a). Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling in the WT/WT control (mean set to 100%; see Methods). **p<0.01, ***p < 0.001; one-way ANOVA with Tukey post hoc test; n = 4-5).


Figure 7HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A before whole cell patch recording in 20 mM Ba2 + . Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of - 80 mV to the indicated potentials ( the voltage protocol is schematized in the upper left corner ) . Representative current traces upon step depolarizations to the reversal potential ( Erev ) for 20 ms to obtain movement of the ON - gating charges ( Qon ) , and subsequent to - 50 mV for 10 ms to obtain tail currents ( Itail ) . Insets : magnifications of exemplary Qon for CaV1 . 2 WT , K1647A , Y1649A , I1654A , F1658A and K1662E . Plots of Itail ( in this panel corrected for variations in cell capacitance ) versus total detectable charge transfer for Qon . Slopes of regression curves are strongly reduced for CaV1 . 2 K1647A , Y1649A and I1654A versus WT ( see Table 7 for more details ) . Dot plots and means + SEM of Qon ( in this panel corrected for variations in cell capacitance ; * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with Bonferroni post - hoc test ; n = 13 - 18 ; see Table 7 for more details ) . The reduction in slope of the regression curve of combined population data for K1647A , Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α - actinin binding to the IQ motif is diminished .

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

Figure 7HEK293 cells were transfected with α11.2, α2δ-1, and β2A before whole cell patch recording in 20 mM Ba2+. Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of -80 mV to the indicated potentials (the voltage protocol is schematized in the upper left corner). Representative current traces upon step depolarizations to the reversal potential (Erev) for 20 ms to obtain movement of the ON-gating charges (Qon), and subsequent to -50 mV for 10 ms to obtain tail currents (Itail). Insets: magnifications of exemplary Qon for CaV1.2 WT, K1647A, Y1649A, I1654A, F1658A and K1662E. Plots of Itail (in this panel corrected for variations in cell capacitance) versus total detectable charge transfer for Qon. Slopes of regression curves are strongly reduced for CaV1.2 K1647A, Y1649A and I1654A versus WT (see Table 7 for more details). Dot plots and means + SEM of Qon (in this panel corrected for variations in cell capacitance; *p<0.05; **p<0.01; one-way ANOVA with Bonferroni post-hoc test; n = 13-18; see Table 7 for more details). The reduction in slope of the regression curve of combined population data for K1647A, Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α-actinin binding to the IQ motif is diminished.


Figure 1 . ( A - D ) EM images of cortical neuropil show an absence of AVs and normal neurite profile in 9 - mo - old NTg mouse brains ( A , arrowheads outline normal neurites ) and a marked accumulation of AVs within enlarged or dystrophic neurites in PS1 / APP mice ( B , arrowheads outline dystrophic neurite profiles in C ) and biopsied brain material from an AD patient ( B , inset ) . At higher magnification ( C ) , AVs include autophagosomes ( arrows ) and multilamellar bodies ( arrowhead ) . In normal dendrites of PS1 / APP mice , multiple AVs are frequently seen ( D , arrows ) . ( E and F ) LC3 quantification analyzed from immunoblots of LC3 - I and LC3 - II ( top ) in prefrontal cortical homogenates from cases of nonaffected ( Cont ) , early stage ( preclinical ) AD ( AD - ES ) , and moderate AD ( AD - MS ; E ) , and from brains of 18 - 22 - mo - old PS1 / APP ( PA ) mice ( n = 3 ; F ) compared with nontransgenic ( NTg ) controls ( n = 3 ; * , P < 0 . 01 ) . Error bars represent SEM . ( G - L ) LC3 immunofluorescence in 9 - mo - old PS1 / APP mice can be seen mainly as puncta in dystrophic dendrites of the cortex ( G , arrows ) and along adjacent dendrites . LC3 ( H , arrows ) is strong in dystrophic neurites in the periphery ( asterisks ) of a thioflavin S - labeled plaque core ( H , inset ) but is less so in neurites closest ( H , arrowheads ) to the Aβ deposit . LC3 is diffuse and uniform in neurons of NTg mice ( I and J ) but is predominantly vesicular and distributed more to the dendrites ( arrows ) than the cell soma ( arrowheads ) in 9 - mo - old PS1 / APP cortex ( K and L ) .

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

Figure 1.(A-D) EM images of cortical neuropil show an absence of AVs and normal neurite profile in 9-mo-old NTg mouse brains (A, arrowheads outline normal neurites) and a marked accumulation of AVs within enlarged or dystrophic neurites in PS1/APP mice (B, arrowheads outline dystrophic neurite profiles in C) and biopsied brain material from an AD patient (B, inset). At higher magnification (C), AVs include autophagosomes (arrows) and multilamellar bodies (arrowhead). In normal dendrites of PS1/APP mice, multiple AVs are frequently seen (D, arrows).(E and F) LC3 quantification analyzed from immunoblots of LC3-I and LC3-II (top) in prefrontal cortical homogenates from cases of nonaffected (Cont), early stage (preclinical) AD (AD-ES), and moderate AD (AD-MS; E), and from brains of 18-22-mo-old PS1/APP (PA) mice (n = 3; F) compared with nontransgenic (NTg) controls (n = 3; *, P < 0.01). Error bars represent SEM.(G-L) LC3 immunofluorescence in 9-mo-old PS1/APP mice can be seen mainly as puncta in dystrophic dendrites of the cortex (G, arrows) and along adjacent dendrites. LC3 (H, arrows) is strong in dystrophic neurites in the periphery (asterisks) of a thioflavin S-labeled plaque core (H, inset) but is less so in neurites closest (H, arrowheads) to the Aβ deposit. LC3 is diffuse and uniform in neurons of NTg mice (I and J) but is predominantly vesicular and distributed more to the dendrites (arrows) than the cell soma (arrowheads) in 9-mo-old PS1/APP cortex (K and L).


Figure 2 . Ultrastructural inspection of brain tissue from PS1 / APP mice ( A - D ) shows that AVs ( A and B , arrows ) are five times more frequent in the dendrites of 8 - wk - old PS1 / APP than in those of age - matched NTg mice . The frequency of AVs per EM field ( C ) and mean number of AVs per EM field ( D ) within the hippocampal molecular layer ( n = 3 ) are shown . LC3immunoblot and analysis ( E ) and immunofluorescent labeling ( F - K ) of the hippocampaldendrites ( brackets ) in 8 - 9 - wk - old PS1 / APP and NTg mice show LC3 - II elevation ( P < 0 . 05 ) in 8 - wk - old PS1 / APP compared with NTg mice ( E ) . ( D ) * , P < 0 . 001 . ( E ) * , P < 0 . 05 . Error bars represent SEM . LC3 immunoreactivity in pyramidal celldendrites is increased in 9 - mo - old ( F - H ) and 9 - wk - old ( I - K ) PS1 / APPmice and frequently exhibits a punctate labeling pattern , which is more evident at 9 mo than at 9 wk ( H and K , arrows ) and is uncommon in NTg mice ( F and I ) . Bars ( F , G , I , and J ) , 20 μm ; ( H and K ) , 10 μm .

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

Figure 2.Ultrastructural inspection of brain tissue from PS1/APP mice (A-D) shows that AVs (A and B, arrows) are five times more frequent in the dendrites of 8-wk-old PS1/APP than in those of age-matched NTg mice. The frequency of AVs per EM field (C) and mean number of AVs per EM field (D) within the hippocampal molecular layer (n = 3) are shown.LC3immunoblot and analysis (E) and immunofluorescent labeling (F-K) of the hippocampaldendrites (brackets) in 8-9-wk-old PS1/APP and NTg mice show LC3-II elevation (P < 0.05) in 8-wk-old PS1/APP compared with NTg mice (E). (D) *, P < 0.001. (E) *, P < 0.05. Error bars represent SEM. LC3 immunoreactivity in pyramidal celldendrites is increased in 9-mo-old (F-H) and 9-wk-old (I-K) PS1/APPmice and frequently exhibits a punctate labeling pattern, which is more evident at 9 mo than at 9 wk (H and K, arrows) and is uncommon in NTg mice (F and I). Bars (F, G, I, and J), 20 μm; (H and K), 10 μm.


Figure 3 . Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1 / APP mice . Cingulate cortex from 9 - mo - old PS1 / APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques ( A ) . At higher magnification , anti - PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona ( B ) . PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1 / APP animals ( C and D ) Arrowheads identify tubulovesicular membrane labeling . PS1 ( C - F , arrows ) was localized on the outer limiting membrane of the AV but not in mitochondria ( Mito ) or on plasma membranes ( PM ) . PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of human brain ( E and F ) by IEM . Arrowheads identify tubulovesicular membrane labeling . PS1 ( C - F , arrows ) was localized on the outer limiting membrane of the AV but not in mitochondria ( Mito ) or on plasma membranes ( PM ) . IEM followed by silver stain enhancement for PS1 was performed on a humanbrain that was diagnosed for AD ( F ) .

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

Figure 3.Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1/APP mice. Cingulate cortex from 9-mo-old PS1/APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques (A). At higher magnification, anti-PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona (B).PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1/APP animals (C and D)Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM).PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of human brain (E and F) by IEM. Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM). IEM followed by silver stain enhancement for PS1 was performed on a humanbrain that was diagnosed for AD (F).


Figure 4 . Induction of macroautophagy in L / APP , SH - SY5Y , and N2a cells . ( A and B ) EM images showing changes in the number of AVs ( arrows ) in L / APP - overexpressing APP695 ( L / APP cells ) grown in complete medium ( A , top left ) or in medium lacking Leu and His ( A , top right ) for 6 h and in SH - SY5Y cells grown in the presence ( A , bottom left ) or absence ( A , bottom right ) of serum . At higher magnification , early and late AVs with typical morphologies are seen in a Leu / His - deprived L / APP cell ( B ) . ( C ) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine ( 0 . 5 μg / ml for 30 min ; left ) and LC3 antibody ( middle , L / APP ; right , SH - SY5Y ) in macroautophagy - induced cells ( bottom ) , which are much less abundant in cells grown in complete medium ( top ) . ( D ) Western blots confirm the cytochemical evidence for increased LC3 - II levels as well as phospho - mTOR ( P - 2481 ) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin ( Rap ) and macroautophagy inhibition by 5 mM 3MA in L / APP , N2a , and SH - SY5Y cells . Immunoblots for LC3 in SH - SY5Y cells and P - 2481 mTOR in L / APP cells have been spliced but are derived from the same blot .

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

Figure 4.Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B).(C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top).(D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.


Figure 5 . Aβ generation in cells after autophagic modulation . Levels of Aβ40 ( A ) , Aβ42 ( B ) , βCTF ( C ) , and APP ( D ) measured by sandwich ELISA after the incubation of L / APP cells ( 6 h ) in conditions that block autophagy ( + Leu , + His , + Leu / + His , and 5 mM 3MA ) , activate macroautophagy ( − Leu , − His , − Leu / − His , and rapamycin ) , or do not affect autophagy ( complete media and enrichment of the deprivation of Gly or Val ) . Values reported as percent difference of control ± SEM ; * , P < 0 . 05 ( at least ) . In similar experiments , Aβ40 and Aβ42 levels from SH - SY5Y ( E and F ) and N2a cells ( G and H ) after various treatments . Error bars represent SEM .

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

Figure 5.Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least).In similar experiments, Aβ40 and Aβ42 levels from SH-SY5Y (E and F) and N2a cells (G and H) after various treatments. Error bars represent SEM.


Figure 6 . Immunolocalization of Aβ in AVs from L / APP cells and PS1 / APPbrains and γ - secretase components ( PS1 and nicastrin ) in L / APP cells . Immunogold localization of Aβ40 ( A ) , Aβ42 ( B ) , PS1 ( C ) , nicastrin ( D ) , and in the absence of primary antibody ( E ) in L / APP cells grown for 6 h in the absence of Leu and His . ( F ) Quantification of gold particle frequency in AV or tubulovesicular compartments ( TBV ) , which comprise 27 . 0 ± 11 . 0 and 19 . 2 ± 7 . 2 % , respectively , of the total cell area . Error bars represent SEM . ( G and H ) IEM followed by silver stain enhancement for Aβ40 was performed in 9 - mo - old PS1 / APP mice .

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

Figure 6.Immunolocalization of Aβ in AVs from L/APP cells and PS1/APPbrains and γ-secretase components (PS1 and nicastrin) in L/APP cells. Immunogold localization of Aβ40 (A), Aβ42 (B), PS1 (C), nicastrin (D), and in the absence of primary antibody (E) in L/APP cells grown for 6 h in the absence of Leu and His. (F) Quantification of gold particle frequency in AV or tubulovesicular compartments (TBV), which comprise 27.0 ± 11.0 and 19.2 ± 7.2%, respectively, of the total cell area. Error bars represent SEM.(G and H) IEM followed by silver stain enhancement for Aβ40 was performed in 9-mo-old PS1/APP mice.


Figure 7 . ( A and B ) Ultrastructure of AVs ( A1 and A2 ) and lysosomes in subcellular fractions from serum - deprived L / APP cells ( A ) and Western blot analysis ( B ) for LC3 - II , rab24 , APP , βCTF , PS1 ( PS1 amino - terminal fragment ) , and nicastrin ( NCT ) in L / APP subcellular fractions . AVs , A1 and A2 ; L , lysosomes ; E , tubulovesicular compartments ( Golgi / ER / endosomes ) ; P , postnuclear pellet ; C , cytosol ; M , mitochondria . Empty lanes in the original blot have been removed from the figure and noted with a white line . ( C ) Rates of cleavage of the fluorogenic substrate in subcellular fractions from L / APP cells grown in − serum media ( left ) or − Leu / − His media ( right ) . PNP , postnuclear pellet . ( D and E ) Proportions of the total recovered cell γ - secretase activity in different subcellular fractions after serum deprivation ( D ) or in uninduced ( + serum ) or induced ( − Leu / − His ) cells ( E ) . Comparison of + serum versus − Leu and − His conditions shows the redistribution of γ - secretase activity from the tubulovesicular to AV fractions after macroautophagic induction . G , Golgi ; A , AV . ( F ) Liquid chromatography mass spectrometry analysis of the fluorogenic substrate after incubation with purified AVs . Selected ion chromatogram corresponding to different cleavage products is displayed . Numbers in graph refer to the cleavage / amino acid site from the Aβ peptide . ( G ) γ - Secretase activity in AV and lysosome fractions from mice blastocysts in which the PS1 and PS2 genes were deleted ( PS KO ; BD8 ) or in which human PS1 was stably transfected into the PS KO blastocysts ( hPS1 ; BD8 / hPS1 ) . Numbers on x axis are in minutes . ( H ) Aβ40 and Aβ42 levels in medium from these cells as detected by sandwich ELISA . Values are given as means ± SEM ( error bars ) .

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

Figure 7.(A and B) Ultrastructure of AVs (A1 and A2) and lysosomes in subcellular fractions from serum-deprived L/APP cells (A) and Western blot analysis (B) for LC3-II, rab24, APP, βCTF, PS1 (PS1 amino-terminal fragment), and nicastrin (NCT) in L/APP subcellular fractions. AVs, A1 and A2; L, lysosomes; E, tubulovesicular compartments (Golgi/ER/endosomes); P, postnuclear pellet; C, cytosol; M, mitochondria. Empty lanes in the original blot have been removed from the figure and noted with a white line.(C) Rates of cleavage of the fluorogenic substrate in subcellular fractions from L/APP cells grown in −serum media (left) or −Leu/−His media (right). PNP, postnuclear pellet.(D and E) Proportions of the total recovered cell γ-secretase activity in different subcellular fractions after serum deprivation (D) or in uninduced (+serum) or induced (−Leu/−His) cells (E). Comparison of +serum versus −Leu and −His conditions shows the redistribution of γ-secretase activity from the tubulovesicular to AV fractions after macroautophagic induction. G, Golgi; A, AV.(F) Liquid chromatography mass spectrometry analysis of the fluorogenic substrate after incubation with purified AVs. Selected ion chromatogram corresponding to different cleavage products is displayed. Numbers in graph refer to the cleavage/amino acid site from the Aβ peptide.(G) γ-Secretase activity in AV and lysosome fractions from mice blastocysts in which the PS1 and PS2 genes were deleted (PS KO; BD8) or in which human PS1 was stably transfected into the PS KO blastocysts (hPS1; BD8/hPS1). Numbers on x axis are in minutes.(H) Aβ40 and Aβ42 levels in medium from these cells as detected by sandwich ELISA. Values are given as means ± SEM (error bars).


Figure 1 EdU - labeled viral DNA forms large foci in ThP1 cells infected by HIV - 1 . EdU images are shown in red , DAPI images in blue . ( A ) Left : nucleus of an infected cell at 48 h p . i . displays three large and bright EdU foci ( colored arrows ) , as well as small and dim punctae ( grey arrows ) . Center : Magnified views have colored borders matching the colors of the arrows . 3D images show that EdU foci are located within the nucleoplasm ( Movie EV1 , Appendix Fig . S2 ) . Right : nucleus of an uninfected cell displays no EdU signal . ( B ) Graph shows EdU intensity profiles along lines shown in A , with colors matching the corresponding arrows . ( C ) Dual - color image of an infected cell with EdU ( red ) and immunolabeled lamin ( grey ) . Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line . The EdU intensity peaks do not coincide with lamin enrichment . See also Fig . EV1A , B . ( D ) Multi - color images of an infected cell showing EdU ( red ) with CA ( yellow ) and integrase ( green ) . The colocalization of nuclear EdU foci with CA and IN ( arrows ) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci . See also Appendix Fig . S3 . ( E , F ) vDNA foci are much larger than the predicted size of single viral genomes . ( E ) Left image shows an observed , EdU - labeled vDNA focus . Right : simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction - limited ( ~ 300 nm resolution ) microscopy . ( F ) Boxplots show the distribution of sizes ( FWHM , full width at half maximum ) of n = 40 measured vDNA foci ( for MOI 100 ) compared to the sizes predicted for linear ( left ) and circular ( right ) chains based on n = 100 simulated configurations each . Blue lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . All differences are highly significant ( Wilcoxon test data vs . model : * * * p ≈ 3 ⋅ 10 − 20 , circular vs . linear model : * * * p ≈ 10 − 17 ) . ( G ) Images of vDNA foci ( arrows ) in ThP1 cells infected with multiplicities of infection ( MOI ) 10 , 50 and 100 . Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots . Scale bars , 3 µm . ( H ) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10 , 50 and 100 . Central lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . Intensities increase significantly with MOI ( Wilcoxon tests : MOI 10 vs . MOI 50 : * * * p ≈ 5 ⋅ 10 − 16 ; MOI 50 vs . MOI 100 : * * * p ≈ 5 ⋅ 10 − 5 ; MOI 10 vs . MOI 100 : * * * p ≈ 2 ⋅ 10 − 23 ) . Data from one experiment . A replicate showed similar results . ( I ) Histograms show the number of foci per nucleus for MOIs of 10 ( blue ) , 50 ( green ) and 100 ( red ) .

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

Figure 1 EdU-labeled viral DNA forms large foci in ThP1 cells infected by HIV-1. EdU images are shown in red, DAPI images in blue. (A) Left: nucleus of an infected cell at 48 h p.i. displays three large and bright EdU foci (colored arrows), as well as small and dim punctae (grey arrows). Center: Magnified views have colored borders matching the colors of the arrows. 3D images show that EdU foci are located within the nucleoplasm (Movie EV1, Appendix Fig. S2). Right: nucleus of an uninfected cell displays no EdU signal. (B) Graph shows EdU intensity profiles along lines shown in A, with colors matching the corresponding arrows.  (C) Dual-color image of an infected cell with EdU (red) and immunolabeled lamin (grey). Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line. The EdU intensity peaks do not coincide with lamin enrichment. See also Fig. EV1A,B.  (D) Multi-color images of an infected cell showing EdU (red) with CA (yellow) and integrase (green). The colocalization of nuclear EdU foci with CA and IN (arrows) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci. See also Appendix Fig. S3. (E,F) vDNA foci are much larger than the predicted size of single viral genomes.  (E) Left image shows an observed, EdU-labeled vDNA focus. Right: simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction-limited (~300 nm resolution) microscopy. (F) Boxplots show the distribution of sizes (FWHM, full width at half maximum) of n=40 measured vDNA foci (for MOI 100) compared to the sizes predicted for linear (left) and circular (right) chains based on n=100 simulated configurations each. Blue lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. All differences are highly significant (Wilcoxon test data vs. model: ***p ≈ 3 ⋅ 10 − 20, circular vs. linear model: ***p ≈ 10 − 17).  (G) Images of vDNA foci (arrows) in ThP1 cells infected with multiplicities of infection (MOI) 10, 50 and 100. Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots. Scale bars, 3 µm.  (H) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10, 50 and 100. Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Intensities increase significantly with MOI (Wilcoxon tests: MOI 10 vs. MOI 50: *** p ≈ 5 ⋅ 10 − 16; MOI 50 vs. MOI 100: ***p ≈ 5 ⋅ 10 − 5; MOI 10 vs. MOI 100: ***p ≈ 2 ⋅ 10 − 23). Data from one experiment. A replicate showed similar results.  (I) Histograms show the number of foci per nucleus for MOIs of 10 (blue), 50 (green) and 100 (red).


Figure 2vDNA foci are also foci of vRNA . ( A ) Dual - color image of an infected ThP1 cell showing the viral DNA ( red ) and the RNA visualized by RNA - FISH ( green ) . Arrows show foci containing both vDNA and vRNA . The bottom row shows the boxed region magnified , with vDNA and vRNA displayed separately and in combination . See also Appendix Fig . S6 . ( C ) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p - value . In ThP1 cells co - infected with a HIV1 - GFP virus and a HIV1 - LUC virus , nuclear foci contain mixtures of both viruses . ( D ) Dual - color image shows RNA - FISH against GFP ( red ) and RNA - FISH against LUC ( green ) . Arrows show foci containing both GFP vRNA and LUC vRNA . The bottom row shows the boxed region magnified , with GFP vRNA and LUC vRNA displayed separately and in combination . See Appendix Fig . S7 . ( F ) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p - value .

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

Figure 2vDNA foci are also foci of vRNA. (A) Dual-color image of an infected ThP1 cell showing the viral DNA (red) and the RNA visualized by RNA-FISH (green). Arrows show foci containing both vDNA and vRNA. The bottom row shows the boxed region magnified, with vDNA and vRNA displayed separately and in combination. See also Appendix Fig. S6.(C) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p-value. In ThP1 cells co-infected with a HIV1-GFP virus and a HIV1-LUC virus, nuclear foci contain mixtures of both viruses. (D) Dual-color image shows RNA-FISH against GFP (red) and RNA-FISH against LUC (green). Arrows show foci containing both GFP vRNA and LUC vRNA. The bottom row shows the boxed region magnified, with GFP vRNA and LUC vRNA displayed separately and in combination. See Appendix Fig. S7.  (F) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p-value.


Figure 3 Images of infected ( right ) or uninfected ( left ) ThP1 cells showing the vDNA ( EdU ) in red , the nucleus ( DAPI ) in blue , and selected nuclear body factors in green . ( A ) Green image shows immunolabeling of CPSF6 . ( B ) Green image shows RNA - FISH against NEAT1 . ( C ) Green image shows immunolabeling of SC35 . See also Appendix Figs . S9 - 14 . ( D ) Boxplots show Pearson correlations r between vDNA ( EdU ) and CPSF6 , NEAT1 or SC35 in 17 - 19 regions of interest ( ROIs ) . Central lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts . Highly significant ( p < 0 . 01 ) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs ; highly significant negative correlations between vDNA and NEAT1 are found in 19 / 19 ROIs , and highly significant positive correlations between vDNA and SC35 are found for 16 / 17 ROIs . Data are from one experiment . For CPSF6 , the experiment was repeated with similar results . ( E ) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above .

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

Figure 3 Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green. (A) Green image shows immunolabeling of CPSF6. (B) Green image shows RNA-FISH against NEAT1. (C) Green image shows immunolabeling of SC35. See also Appendix Figs. S9-14.  (D) Boxplots show Pearson correlations r between vDNA (EdU) and CPSF6, NEAT1 or SC35 in 17-19 regions of interest (ROIs). Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts. Highly significant (p<0.01) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs; highly significant negative correlations between vDNA and NEAT1 are found in 19/19 ROIs, and highly significant positive correlations between vDNA and SC35 are found for 16/17 ROIs. Data are from one experiment. For CPSF6, the experiment was repeated with similar results.  (E) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above.


Figure 4 ( A ) Image of a ThP1 cell infected with an integration - deficient HIV - 1 carrying the mutation D116A in the catalytic site of IN . The EdU - labeled vDNA is shown in red and the vRNA detected by RNA - FISH in green . The nucleus ( DAPI ) is shown in blue . See Appendix Fig . S12 for a larger image region . ( C ) Scatter plot shows vDNA and vRNA intensities in colocalizing vDNA and vRNA clusters with the Spearman correlation r and associated p - value . ( D ) ThP1 cells were infected with HIV - 1 at an MOI of 20 , in absence ( left ) or presence ( right ) of nevirapine ( NVP , 10 µM ) . Blue : DAPI staining . Green : vRNA ( RNA - FISH ) . Red : vDNA ( EdU ) . ( E ) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters . ( F ) Image of a ThP1 cell infected by HIV - 1 with EU labeled RNA in absence ( left ) or presence ( right ) of nevirapine ( NVP , 10 µM ) . Blue : DAPI staining . Green : vRNA ( RNA - FISH ) . Grey : EU - labeled vRNA . See Appendix Fig . S19 for a larger image region . ( G ) Intensity profiles of EU and RNA - FISH along the dotted lines in panel F ( top ) .

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

Figure 4 (A) Image of a ThP1 cell infected with an integration-deficient HIV-1 carrying the mutation D116A in the catalytic site of IN. The EdU-labeled vDNA is shown in red and the vRNA detected by RNA-FISH in green. The nucleus (DAPI) is shown in blue. See Appendix Fig. S12 for a larger image region.  (C) Scatter plot shows vDNA and vRNA intensities in colocalizing vDNA and vRNA clusters with the Spearman correlation r and associated p-value.  (D) ThP1 cells were infected with HIV-1 at an MOI of 20, in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Red: vDNA (EdU). (E) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters.  (F) Image of a ThP1 cell infected by HIV-1 with EU labeled RNA in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Grey: EU-labeled vRNA. See Appendix Fig. S19 for a larger image region. (G) Intensity profiles of EU and RNA-FISH along the dotted lines in panel F (top).


Figure 5 The left panel shows the timeline of drug exposure experiments . ( A - D ) Images show vRNA and vDNA in an infected ThP1 cell ( MOI 20 ) for each of four experimental conditions . See Appendix Fig . S15 - S17 for images of larger regions . ( A , B ) ThP1 cells were exposed to NVP for 48 h ( A ) or 72 h after infection ( B ) , then NVP was washed out , and cells were fixed for click chemistry 24 h later . ( C ) Cells were exposed to PF74 for 72 h after infection , then fixed for click chemistry . ( D ) Cells were exposed to NVP for 24 h after infection , then NVP was washed out , and cells were exposed to PF74 , before being fixed for click chemistry 24 h later . ( E , F , H ) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p - values . Replicates of the four experiments yielded similar results . ( G ) qPCR measures DNA synthesis ( left ) and nuclear import ( right ) at 72 h p . i . in absence of drug treatment , or after exposure to NVP or PF74 . Bars and error bars define mean and standard deviation , respectively . Samples were analyzed in triplicate and two biological replicates were performed .

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

Figure 5 The left panel shows the timeline of drug exposure experiments. (A-D) Images show vRNA and vDNA in an infected ThP1 cell (MOI 20) for each of four experimental conditions. See Appendix Fig. S15-S17 for images of larger regions. (A,B) ThP1 cells were exposed to NVP for 48 h (A) or 72 h after infection (B), then NVP was washed out, and cells were fixed for click chemistry 24 h later. (C) Cells were exposed to PF74 for 72 h after infection, then fixed for click chemistry. (D) Cells were exposed to NVP for 24 h after infection, then NVP was washed out, and cells were exposed to PF74, before being fixed for click chemistry 24 h later. (E,F,H) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p-values. Replicates of the four experiments yielded similar results. (G) qPCR measures DNA synthesis (left) and nuclear import (right) at 72 h p.i. in absence of drug treatment, or after exposure to NVP or PF74. Bars and error bars define mean and standard deviation, respectively. Samples were analyzed in triplicate and two biological replicates were performed.


Figure 6 The left panel shows the timeline of infection and / or drug exposure experiments . ( A - D ) Percentage of GFP positive cells at 3 and 7 days post - infection analyzed by FACS in absence of EdU . ( A ) Uninfected control cells . ( B ) Untreated , infected ThP1 cells fixed at 3 d ( 72 h ) p . i . ( C ) Infected cells were exposed to NVP for 72 h , then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p . i . ( D ) Infected cells were cultured for 72 h p . i . , then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p . i . ( E - H ) Multicolor images of ThP1 cells infected with a GFP - reporter virus . vRNA ( RNA - FISH ) is labeled in red , GFP in green , and nuclei ( DAPI ) in blue . ( E ) Uninfected control cells . ( F ) Untreated , infected ThP1 cells fixed at 3 d ( 72 h ) p . i . ( G ) Infected cells were exposed to NVP for 72 h , then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p . i . ( H ) Infected cells were cultured for 72 h p . i . , then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p . i . The experiment was repeated three times .

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

Figure 6 The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Percentage of GFP positive cells at 3 and 7 days post-infection analyzed by FACS in absence of EdU. (A) Uninfected control cells. (B) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (C) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (D) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. (E-H) Multicolor images of ThP1 cells infected with a GFP-reporter virus. vRNA (RNA-FISH) is labeled in red, GFP in green, and nuclei (DAPI) in blue. (E) Uninfected control cells. (F) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (G) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (H) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. The experiment was repeated three times.


Figure 7 The left panel shows the timeline of infection and / or drug exposure experiments . ( A - D ) Multicolor images of vDNA , vRNA , DAPI and / or GFP in monocyte derived macrophages ( MDMs ) from two different donors , infected with HIV1 . Cells from donor 1 were infected with VSV - G - pseudotyped HIV - 1ΔEnv Vpx carrying a GFP reporter ( A ) and cells from donor 2 were infected with a VSV - G - pseudotyped HIV - 1ΔEnv without Vpx ( B - D ) . ( A , B ) Infected cells were left untreated and fixed at 6 d p . i . ( C ) Infected cells were treated with 10 µM Nevirapine ( NVP ) throughout the experiment and fixed at 6 d p . i . ( D ) Infected cells were exposed to NVP for 3 d p . i . , then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p . i . Arrows in ( A - D ) show the position of selected vRNA foci . ( E ) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p . i . ( B ) , measured by the FWHM of n = 24 foci . The red line defines the median , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . ( F ) Boxplot shows Pearson ' s r , measuring colocalization of DNA with RNA foci in the same cells ( n = 20 ) . Center line defines the median , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . ( G ) FACS analysis of Gag positive cells from donor 2 in different conditions . Red : uninfected cells . Blue : untreated infected cells at 7 d p . i . as in B . Orange : cells were treated with NVP for 7 d as in C . Green : infected cells were exposed to NVP for 3 d , then NVP was washed out and cells were cultured for another 4 d as in D . Median of fluorescence intensities ( MFI ) for each sample are shown in the table .

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

Figure 7 The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Multicolor images of vDNA, vRNA, DAPI and/or GFP in monocyte derived macrophages (MDMs) from two different donors, infected with HIV1. Cells from donor 1 were infected with VSV-G-pseudotyped HIV-1ΔEnv Vpx carrying a GFP reporter (A) and cells from donor 2 were infected with a VSV-G-pseudotyped HIV-1ΔEnv without Vpx (B-D). (A,B) Infected cells were left untreated and fixed at 6 d p.i. (C) Infected cells were treated with 10 µM Nevirapine (NVP) throughout the experiment and fixed at 6 d p.i. (D) Infected cells were exposed to NVP for 3 d p.i., then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p.i. Arrows in (A-D) show the position of selected vRNA foci. (E) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p.i. (B), measured by the FWHM of n=24 foci. The red line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. (F) Boxplot shows Pearson's r, measuring colocalization of DNA with RNA foci in the same cells (n=20). Center line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points.  (G) FACS analysis of Gag positive cells from donor 2 in different conditions. Red: uninfected cells. Blue: untreated infected cells at 7 d p.i. as in B. Orange: cells were treated with NVP for 7 d as in C. Green: infected cells were exposed to NVP for 3 d, then NVP was washed out and cells were cultured for another 4 d as in D. Median of fluorescence intensities (MFI) for each sample are shown in the table.


Figure 1Macrophages were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( A ) Immunoblot of co - immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β - actin as loading control . ( B ) Cells were incubated with 10 µg / mL pepstatin A for 4 h prior to lysis . Immunoblots of LC3B isoforms using antibody to LC3B or β - actin . ( C ) Cells were fixed and permeabilized then stained with antibody to LC3B ( green ) and Hoechst 33342 ( blue ) . Scale bars indicate 10 µm . Left , representative fluorescence microscopy images . When puncta are not present , LC3B staining gives rise to a diffuse cytoplasmic pattern . Right , percentage of cells with LC3B puncta . Bar charts are presented as the means ± s . e . m . , n = 6 . ( D ) Immunoblots of SQSTM1 using antibody to SQSTM1 or β - actin . * p < 0 . 05 .

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

Figure 1Macrophages were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Immunoblot of co-immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β-actin as loading control.(B) Cells were incubated with 10 µg/mL pepstatin A for 4 h prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 6.(D) Immunoblots of SQSTM1 using antibody to SQSTM1 or β-actin. * p<0.05.