figf5 ( a ) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation . At 7 weeks : HdhHD / + Dnchc1Loa / + n = 9 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 14 . At 9 weeks : HdhHD / + Dnchc1Loa / + n = 8 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 027 . At 11 weeks : HdhHD / + Dnchc1Loa / + n = 6 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 001 . At 13 weeks : HdhHD / + Dnchc1Loa / + n = 5 , HdhHD / + Dnchc1 + / + n = 8 , P = 0 . 021 . At 15 weeks : HdhHD / + Dnchc1Loa / + n = 3 , HdhHD / + Dnchc1 + / + n = 5 , P = 0 . 021 . ( b , c ) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease . HdhHD / + Dnchc1Loa / + , HdhHD / + Dnchc1 + / + and Hdh + / + Dnchc1Loa / + mice are shown . An average of six mice were used at every time point , with at least three mice per group after 13 weeks . ( b ) Grip strength . Overall effect for all time points between the three genotypes : P = 0 . 0024 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and HdhHD / + Dnchc1 + / + : P = 0 . 001 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and Hdh + / + Dnchc1Loa / + : P = 0 . 0157 . Effect at each time point between the three genotypes : 7 weeks , P = 0 . 023 ; 9 weeks , P = 0 . 001 ; 11 weeks , P 0 . 0001 ; 13 weeks , P = 0 . 004 ; 15 weeks , P = 0 . 336 ( most of the double mutants were dead by 15 weeks ) . ( c ) Accelerating rotarod . Overall effect for all time points between the three genotypes : P = 0 . 0367 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and HdhHD / + Dnchc1 + / + : P = 0 . 0245 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and Hdh + / + Dnchc1Loa / + : P = 0 . 0553 . Effect at each time point between the three genotypes : 8 weeks , P 0 . 001 ; 10 weeks , P = 0 . 005 ; 12 weeks , P = 0 . 001 ; 14 weeks , P = 0 . 009 ; 16 weeks , P = 0 . 039 . ( d ) Reduced lifespan of HdhHD / + Dnchc1Loa / + mice . HdhHD / + Dnchc1Loa / + ( red line ) n = 13 , HdhHD / + Dnchc1 + / + ( green line ) n = 9 , P = 0 . 038 . Mice were subjected to humane end points , and so the survival curve reflects the onset of the end points rather than the complete lifespan . Asterisks ( * ) represent mice that were found dead without reaching humane end points . No mortality of Hdh + / + Dnchc1Loa / + mice was observed .

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

figf5(a) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation. At 7 weeks: HdhHD/+ Dnchc1Loa/+ n = 9, HdhHD/+ Dnchc1+/+ n = 9, P = 0.14. At 9 weeks: HdhHD/+ Dnchc1Loa/+ n = 8, HdhHD/+ Dnchc1+/+ n = 9, P = 0.027. At 11 weeks: HdhHD/+ Dnchc1Loa/+ n = 6, HdhHD/+ Dnchc1+/+ n = 9, P = 0.001. At 13 weeks: HdhHD/+ Dnchc1Loa/+ n = 5, HdhHD/+ Dnchc1+/+ n = 8, P = 0.021. At 15 weeks: HdhHD/+ Dnchc1Loa/+ n = 3, HdhHD/+ Dnchc1+/+ n = 5, P = 0.021.(b,c) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease. HdhHD/+ Dnchc1Loa/+, HdhHD/+ Dnchc1+/+ and Hdh+/+ Dnchc1Loa/+ mice are shown. An average of six mice were used at every time point, with at least three mice per group after 13 weeks. (b) Grip strength. Overall effect for all time points between the three genotypes: P = 0.0024. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.001. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0157. Effect at each time point between the three genotypes: 7 weeks, P = 0.023; 9 weeks, P = 0.001; 11 weeks, P 0.0001; 13 weeks, P = 0.004; 15 weeks, P = 0.336 (most of the double mutants were dead by 15 weeks). (c) Accelerating rotarod. Overall effect for all time points between the three genotypes: P = 0.0367. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.0245. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0553. Effect at each time point between the three genotypes: 8 weeks, P 0.001; 10 weeks, P = 0.005; 12 weeks, P = 0.001; 14 weeks, P = 0.009; 16 weeks, P = 0.039.(d) Reduced lifespan of HdhHD/+ Dnchc1Loa/+ mice. HdhHD/+ Dnchc1Loa/+ (red line) n = 13, HdhHD/+ Dnchc1+/+ (green line) n = 9, P = 0.038. Mice were subjected to humane end points, and so the survival curve reflects the onset of the end points rather than the complete lifespan. Asterisks (*) represent mice that were found dead without reaching humane end points. No mortality of Hdh+/+ Dnchc1Loa/+ mice was observed.


figf6 ( a ) Brain sections from HdhHD / + Dnchc1 + / + or HdhHD / + Dnchc1Loa / + mice were stained with antibody to huntingtin ( EM48 ; green ) . No EM48 - stained aggregates were detected in brains of Hdh + / + Dnchc1Loa / + mice . Arrows indicate cells with aggregates . ( b ) Western - blot analysis of mouse brain lysates from wild - type ( WT ) or Loa mutant mice using antibodies to LC3 and to actin .

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 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

figf6(a) Brain sections from HdhHD/+ Dnchc1+/+ or HdhHD/+ Dnchc1Loa/+ mice were stained with antibody to huntingtin (EM48; green). No EM48-stained aggregates were detected in brains of Hdh+/+ Dnchc1Loa/+ mice. Arrows indicate cells with aggregates.(b) Western-blot analysis of mouse brain lysates from wild-type (WT) or Loa mutant mice using antibodies to LC3 and to actin.


Figure 1 . ( A ) HeLa cells were fixed and stained for p62 . The different patterns of p62 distribution were scored in > 200 cells as cytoplasmic with small bodies , cytoplasmic with large and intense bodies , equal nuclear and cytoplasmic staining , and nuclear enriched . The percentage of cells in the respective groups is indicated . ( B ) Expression level of GFP - p62 in HeLa cells stably expressing GFP - p62 ( S - GFP - p62 ) compared with the level of endogenous p62 in the parent HeLa cells . ( C ) Cells stably expressing GFP - p62 display a cytoplasmic punctuate localization of GFP - tagged p62 similar to endogenous p62 . Video microscopy of live cells demonstrated that the small , faint bodies ( thin arrows ) displayed directed migration , whereas the majority of the larger , intense bodies ( thick arrows ) were nonmigratory ( Videos 1 and 2 , available at http : / / www . jcb . org / cgi / content / full / jcb . 200507002 / DC1 ) . A still image from Video 1 is shown here . ( D ) Bodies containing either endogenous p62 or transiently or stably transfected GFP - p62 all contain polyubiquitin . HeLa cells were fixed and stained with p62 and polyubiquitin ( clone FK1 ) mAbs directly coupled with AlexaFluor555 ( red ) and AlexaFluor488 ( green ) , respectively . Cells expressing GFP - p62 were only stained for polyubiquitin ( red ) . Bars , 20 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1.(A) HeLa cells were fixed and stained for p62. The different patterns of p62 distribution were scored in >200 cells as cytoplasmic with small bodies, cytoplasmic with large and intense bodies, equal nuclear and cytoplasmic staining, and nuclear enriched. The percentage of cells in the respective groups is indicated.(B) Expression level of GFP-p62 in HeLa cells stably expressing GFP-p62 (S-GFP-p62) compared with the level of endogenous p62 in the parent HeLa cells.(C) Cells stably expressing GFP-p62 display a cytoplasmic punctuate localization of GFP-tagged p62 similar to endogenous p62. Video microscopy of live cells demonstrated that the small, faint bodies (thin arrows) displayed directed migration, whereas the majority of the larger, intense bodies (thick arrows) were nonmigratory (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1). A still image from Video 1 is shown here.(D) Bodies containing either endogenous p62 or transiently or stably transfected GFP-p62 all contain polyubiquitin. HeLa cells were fixed and stained with p62 and polyubiquitin (clone FK1) mAbs directly coupled with AlexaFluor555 (red) and AlexaFluor488 (green), respectively. Cells expressing GFP-p62 were only stained for polyubiquitin (red). Bars, 20 μm.


Figure 2 . ( A ) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts . Asterisks indicate point mutations in the PB1 ( D69A ) and UBA ( I431A ) domains , respectively . ( B ) S - GFP - p62 cells were transiently transfected with the indicated myc - tagged p62 constructs , fixed , and stained for the myc tag . Bars , 20 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 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

Figure 2.(A) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts. Asterisks indicate point mutations in the PB1 (D69A) and UBA (I431A) domains, respectively.(B) S-GFP-p62 cells were transiently transfected with the indicated myc-tagged p62 constructs, fixed, and stained for the myc tag. Bars, 20 μm.


Figure 3 . ( A ) Localization of bodies containing endogenous p62 or transiently expressed GFP - p62 relative to EEA1 - positive early endosomes . Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488 , and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555 . Alternatively , transiently expressed GFP - p62 was expressed in HeLa cells , and EEA1 was stained red with EEA1 mAb ( bottom ) . The boxed area is shown to the right at a higher magnification . ( B ) Colocalization of GFP - p62 and CD63 ( stained red using a mAb ) in HeLa cells stably expressing GFP - p62 . ( C ) Immunoelectron micrograph of S - GFP - p62 stained with a GFP pAb ( 10 - nm gold particle , arrows ) and monoclonal CD63 ( 15 - nm gold particle , arrowheads ) . ( D ) Rapid detergent extraction of GFP - p62 from LysoTracker - positive acidic organelles . HeLa cells transiently expressing GFP - p62 were labeled with LysoTracker for 60 min . The detergent extractions were imaged in a time series with 15 - s time intervals after adding 1 % Triton X - 100 . The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction . Bars ( A , B , and D ) , 20 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 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 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

Figure 3.(A) Localization of bodies containing endogenous p62 or transiently expressed GFP-p62 relative to EEA1-positive early endosomes. Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488, and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555. Alternatively, transiently expressed GFP-p62 was expressed in HeLa cells, and EEA1 was stained red with EEA1 mAb (bottom). The boxed area is shown to the right at a higher magnification.(B) Colocalization of GFP-p62 and CD63 (stained red using a mAb) in HeLa cells stably expressing GFP-p62.(C) Immunoelectron micrograph of S-GFP-p62 stained with a GFP pAb (10-nm gold particle, arrows) and monoclonal CD63 (15-nm gold particle, arrowheads).(D) Rapid detergent extraction of GFP-p62 from LysoTracker-positive acidic organelles. HeLa cells transiently expressing GFP-p62 were labeled with LysoTracker for 60 min. The detergent extractions were imaged in a time series with 15-s time intervals after adding 1% Triton X-100. The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction. Bars (A, B, and D), 20 μm.


Figure 4 . ( A ) Immuno - EM of GFP - p62 . S - GFP - p62 cells were labeled with rabbit anti - GFP ( Abcam ) followed by protein A - gold ( 15 nm ) . We observed labeling in membrane - free cytosolic structures and sequestosomes ( arrows ) as well as in endosomes ( Endo ) . ( B ) Correlative immunofluorescence / EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes . The insets show two magnifications of the sequestosome , which is labeled 1 . ( C ) Representative image of a p62 - containing autophagosome . HeLa cells transfected with GFP - p62 were immunogold labeled as in A . Note the cisternal - like membrane ( arrowheads ) surrounding the GFP - positive material . The arrow indicates a fused endosome .

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

Figure 4.(A) Immuno-EM of GFP-p62. S-GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A-gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo).(B) Correlative immunofluorescence/EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes. The insets show two magnifications of the sequestosome, which is labeled 1.(C) Representative image of a p62-containing autophagosome. HeLa cells transfected with GFP-p62 were immunogold labeled as in A. Note the cisternal-like membrane (arrowheads) surrounding the GFP-positive material. The arrow indicates a fused endosome.


Figure 5 . ( A ) Comparison of p62 degradation using pulse - chase labeling with 35S - methionine and immunoblotting after cycloheximide ( CHX ) treatment . HeLa cells were pulsed with 35S - methionine and incubated for the indicated times in nonradioactive medium . After immunopurification , the amount of radioactive p62 was determined by autoradiography , and the total amount of p62 was determined by an immunoblot of the same membrane . The p62 level in total cellular lysates after different times of 10 μg / ml cycloheximide treatment determined by immunoblotting ( bottom ) . ( B ) The levels of p62 and LC3 - II change with autophagic activity . HeLa or S - GFP - p62 cells were either left untreated or rapamycin ( 10 μg / ml ) or bafilomycin A1 ( Baf . A1 ; 10 μg / ml ) was added for 18 h . Immunoblots were sequentially probed using LC3 , p62 , and actin antibodies . ( C and D ) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy . HeLa cells or S - GFP - p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h , the cells were fixed , and p62 was either stained red using a p62 mAb ( C ) or imaged directly ( D ) . Nuclei were visualized using the Draq5 DNA stain . The settings for imaging were identical for the treated cells and the untreated control . ( E ) The majority of S - GFP - p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3 . S - GFP - p62 cells were transiently transfected with myc - LC3 . Myc - LC3 was stained red using an anti - myc tag mAb . The boxed area indicates the part of the cell that is shown to the left at a higher magnification . Bars , 5 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 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 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

Figure 5.(A) Comparison of p62 degradation using pulse-chase labeling with 35S-methionine and immunoblotting after cycloheximide (CHX) treatment. HeLa cells were pulsed with 35S-methionine and incubated for the indicated times in nonradioactive medium. After immunopurification, the amount of radioactive p62 was determined by autoradiography, and the total amount of p62 was determined by an immunoblot of the same membrane. The p62 level in total cellular lysates after different times of 10 μg/ml cycloheximide treatment determined by immunoblotting (bottom).(B) The levels of p62 and LC3-II change with autophagic activity. HeLa or S-GFP-p62 cells were either left untreated or rapamycin (10 μg/ml) or bafilomycin A1 (Baf. A1; 10 μg/ml) was added for 18 h. Immunoblots were sequentially probed using LC3, p62, and actin antibodies.(C and D) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy. HeLa cells or S-GFP-p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h, the cells were fixed, and p62 was either stained red using a p62 mAb (C) or imaged directly (D). Nuclei were visualized using the Draq5 DNA stain. The settings for imaging were identical for the treated cells and the untreated control.(E) The majority of S-GFP-p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3. S-GFP-p62 cells were transiently transfected with myc-LC3. Myc-LC3 was stained red using an anti-myc tag mAb. The boxed area indicates the part of the cell that is shown to the left at a higher magnification. Bars, 5 μm.


Figure 6 . ( A ) HeLa cells transiently transfected with GFP - LC3 alone or cotransfected with siRNA against p62 , HA - p62 , or HA - p62 D69A were either left in normal medium or starved for amino acids for 1 h . The cells were fixed , and p62 was stained using a p62 mAb . More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP - LC3 as either diffuse or punctuate . The frequency of GFP - LC3 - positive cells with punctuate localization are indicated to the right . ( B ) Endogenous p62 as well as coexpressed myc - tagged wild - type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP - LC3 from HeLa cell extracts . GFP or GFP - LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs . The cell cultures were either left untreated or starved for amino acids for 1 h as indicated . Copurified endogenous or ectopically expressed myc - tagged p62 constructs were detected using the p62 mAb . Bars , 20 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 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

Figure 6.(A) HeLa cells transiently transfected with GFP-LC3 alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A were either left in normal medium or starved for amino acids for 1 h. The cells were fixed, and p62 was stained using a p62 mAb. More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-LC3-positive cells with punctuate localization are indicated to the right.(B) Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs. The cell cultures were either left untreated or starved for amino acids for 1 h as indicated. Copurified endogenous or ectopically expressed myc-tagged p62 constructs were detected using the p62 mAb. Bars, 20 μm.


Figure 7 . ( A ) HeLa cells transiently expressing a Flag - tagged NH2 - terminal fragment ( amino acids 1 - 171 ) of huntingtin containing a 68 - polyglutamine expansion , Flag - N - HttQ68 . After 48 h , the cells were fixed , and Flag - N - HttQ68 was stained green using a Flag mAb . Endogenous p62 was stained red using the p62 pAb . ( B ) Endogenous p62 forms a shell around aggregated GFP - N - HttQ68 . Transiently transfected HeLa cells were fixed 48 h after transfection , and endogenous p62 was detected using a p62 mAb ( red ) . ( C and D ) The p62 shell surrounding GFP - N - HttQ68 was detected independently of the use of antibodies . ( C ) GFP - N - HttQ68 and DsRed - tagged p62 were cotransfected into HeLa cells , and images of live cells were obtained after 48 h of expression . The left panel shows one confocal plane of a representative GFP - N - HttQ68 and DsRed - p62 double - positive structure , and the right panel shows the Z - stack of planes with side views of the aggregate at the side and at the top . N , nucleus . ( A - C ) Nuclei were detected using Draq5 . ( D ) GFP - N - HttQ68 and tdTomato - tagged p62 were cotransfected into HeLa cells , and images of live cells were obtained after 24 h of expression . ( E ) Transiently expressed Flag - N - HttQ68 and myc - LC3 colocalize in GFP - p62 - positive structures . S - GFP - p62 cells were fixed 48 h after transfection , Flag - N - HttQ68 was stained red using a Flag tag mAb , and myc - LC3 was stained far red ( blue ) using a chicken myc tag pAb ( top ) . The bottom panel shows a live cell image of a GFP - N - HttQ68 aggregate surrounded by a shell containing tdTomato - LC3 . Bars , 5 μm .

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Figure 7.(A) HeLa cells transiently expressing a Flag-tagged NH2-terminal fragment (amino acids 1-171) of huntingtin containing a 68-polyglutamine expansion, Flag-N-HttQ68. After 48 h, the cells were fixed, and Flag-N-HttQ68 was stained green using a Flag mAb. Endogenous p62 was stained red using the p62 pAb.(B) Endogenous p62 forms a shell around aggregated GFP-N-HttQ68. Transiently transfected HeLa cells were fixed 48 h after transfection, and endogenous p62 was detected using a p62 mAb (red).(C and D) The p62 shell surrounding GFP-N-HttQ68 was detected independently of the use of antibodies. (C) GFP-N-HttQ68 and DsRed-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 48 h of expression. The left panel shows one confocal plane of a representative GFP-N-HttQ68 and DsRed-p62 double-positive structure, and the right panel shows the Z-stack of planes with side views of the aggregate at the side and at the top. N, nucleus. (A-C) Nuclei were detected using Draq5. (D) GFP-N-HttQ68 and tdTomato-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 24 h of expression.(E) Transiently expressed Flag-N-HttQ68 and myc-LC3 colocalize in GFP-p62-positive structures. S-GFP-p62 cells were fixed 48 h after transfection, Flag-N-HttQ68 was stained red using a Flag tag mAb, and myc-LC3 was stained far red (blue) using a chicken myc tag pAb (top). The bottom panel shows a live cell image of a GFP-N-HttQ68 aggregate surrounded by a shell containing tdTomato-LC3. Bars, 5 μm.


Figure 8 . ( A ) GFP - positive cells were scored as live or apoptotic by the appearance of nuclear Draq5 staining . After 48 h of expression , GFP - positive cells attached to the surface with a normal nuclear appearance ( open arrowheads ) and were scored as live , whereas rounded , partially detached cells with a condensed or fragmented nuclei ( arrows ) were scored as dead . More than 200 GFP - positive cells in randomly selected microscope views were scored . Bars , 20 μm . ( B ) The effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking the UBA domain on N - HttQ68 - induced cell death in HeLa and SHSY - 5Y neuroblastoma cells .

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Figure 8.(A) GFP-positive cells were scored as live or apoptotic by the appearance of nuclear Draq5 staining. After 48 h of expression, GFP-positive cells attached to the surface with a normal nuclear appearance (open arrowheads) and were scored as live, whereas rounded, partially detached cells with a condensed or fragmented nuclei (arrows) were scored as dead. More than 200 GFP-positive cells in randomly selected microscope views were scored. Bars, 20 μm.(B) The effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking the UBA domain on N-HttQ68-induced cell death in HeLa and SHSY-5Y neuroblastoma cells.


Fig . 1Overall structure of 2019 - nCoV RBD bound with ACE2 . ACE2 is colored green . 2019 - nCoV RBD core is colored cyan and RBM is colored red . Disulfide bonds in the 2019 - nCoV RBD are shown as stick and indicated by yellow arrows . The N - terminal helix of ACE2 responsible for binding is labeled .

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Fig. 1Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.


Fig . 2Alignment of the 2019 - nCoV RBD ( core in cyan and RBM in red ) and SARS - CoV RBD ( core in orange and RBM in blue ) structures . Structural alignment of 2019 - nCoV RBD / ACE2 and SARS - CoV RBD / ACE2 complexes . 2019 - nCoV RBD is colored cyan and red , its interacting ACE2 is colored green . SARS - CoV RBD is colored orange and blue , its interacting ACE2 is colored salmon . The PDB code for SARS - CoV RBD / ACE2 complex : 2AJF .

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Fig. 2Alignment of the 2019-nCoV RBD (core in cyan and RBM in red) and SARS-CoV RBD (core in orange and RBM in blue) structures.Structural alignment of 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 complexes. 2019-nCoV RBD is colored cyan and red, its interacting ACE2 is colored green. SARS-CoV RBD is colored orange and blue, its interacting ACE2 is colored salmon. The PDB code for SARS-CoV RBD/ACE2 complex: 2AJF.


Fig . 3Contacting residues shown as stick at the 2019 - nCoV RBD / ACE2 and SARS - CoV RBD / ACE2 interfaces . Positions in both RBDs involved in ACE2 binding are indicated by red labels .

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Fig. 3Contacting residues shown as stick at the 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 interfaces. Positions in both RBDs involved in ACE2 binding are indicated by red labels.


Fig 1A . In response to a 1 s light pulse , cac alleles lack on transients at day 3 and day 33 . B . Quantification of depolarization amplitudes ( top ) and on amplitudes ( bottom ) . Data are presented as means ± standard error of the mean ( SEM ) .

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Fig 1A. In response to a 1 s light pulse, cac alleles lack on transients at day 3 and day 33.B. Quantification of depolarization amplitudes (top) and on amplitudes (bottom). Data are presented as means ± standard error of the mean (SEM).


Fig 2A . TEM sections of 3 - and 27 - days - old photoreceptor terminals of flies . Terminals of 27 - days - old flies show terminal expansion , loss of capitate projections ( CPs ) and active zones ( AZs ) and increased number of mitochondria per terminal . Scale bars , 500 nm . B . Inset from A representing the photoreceptor terminal boxed in white . Yellow arrow indicates double - membraned autophagosome , red indicates amphisome , and blue indicates two fusion - primed AVs . Scale bars , 500 nm . C - E . Quantification of CPs , AZs , and mitochondria per cartridge , respectively . Data are presented as means ± SEM . F . Quantification of the different AVs per cartridge . The mutant terminals accumulate late stage AVs and fusion - primed AVs ( ns : not significant ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ) . Data are presented as means ± SEMG . 30 - days old fly eye brain complexes stained with neuronal marker Elav ( blue ) and poly - Ub antibody ( red ) . The right panel is the enlarged images of the boxed regions at the left sideH . The level of p62 is increased in cacJ brain . I . Quantification of H . Data are presented as means ± SEM . ( * : p < 0 . 05 ) .

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Fig 2A. TEM sections of 3- and 27-days-old photoreceptor terminals of flies. Terminals of 27-days-old flies show terminal expansion, loss of capitate projections (CPs) and active zones (AZs) and increased number of mitochondria per terminal. Scale bars, 500 nm.B. Inset from A representing the photoreceptor terminal boxed in white. Yellow arrow indicates double-membraned autophagosome, red indicates amphisome, and blue indicates two fusion-primed AVs. Scale bars, 500 nm.C-E. Quantification of CPs, AZs, and mitochondria per cartridge, respectively. Data are presented as means ± SEM.F. Quantification of the different AVs per cartridge. The mutant terminals accumulate late stage AVs and fusion-primed AVs (ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). Data are presented as means ± SEMG. 30-days old fly eye brain complexes stained with neuronal marker Elav (blue) and poly-Ub antibody (red). The right panel is the enlarged images of the boxed regions at the left sideH. The level of p62 is increased in cacJ brain. I. Quantification of H. Data are presented as means ± SEM. (*: p < 0.05).


Fig 3Autophagy defects observed in cac mutants are not due to defects in neurotransmitter release , but due to defects in lysosomal fusion . TEM sections of 1 - day - old and 30 - days - old photoreceptor terminals of flies raised in 12 h light / dark conditions . A , B , D , and E . stj and Vha100 - 1 ( V0 ) mutant terminals accumulate AVs upon aging . G , H . n - Syb mutant photoreceptor terminals show increased SVs , but not a significant accumulation of AVs and fusion - primed AVs . J , K , M and N . Vamp7 and fab1 mutant photoreceptor terminals show the highest increase in AV accumulation . C , F , I , L , O . Quantification of the different AVs for each genotype ( * * : p < 0 . 01 ) . Eyes from three animals for each genotype at each age were analyzed and the counted cartridge number were listed as " n = " in the figure . Data are presented as means ± standard deviation ( SD ) . Scale bars , 1 μm .

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Fig 3Autophagy defects observed in cac mutants are not due to defects in neurotransmitter release, but due to defects in lysosomal fusion.TEM sections of 1-day-old and 30-days-old photoreceptor terminals of flies raised in 12 h light/dark conditions. A, B, D, and E. stj and Vha100-1 (V0) mutant terminals accumulate AVs upon aging. G, H. n-Syb mutant photoreceptor terminals show increased SVs, but not a significant accumulation of AVs and fusion-primed AVs. J, K, M and N. Vamp7 and fab1 mutant photoreceptor terminals show the highest increase in AV accumulation. C, F, I, L, O. Quantification of the different AVs for each genotype (**: p < 0.01). Eyes from three animals for each genotype at each age were analyzed and the counted cartridge number were listed as "n = " in the figure. Data are presented as means ± standard deviation (SD). Scale bars, 1 μm.


Fig 4A . Calbindin and hematoxylin staining of mice cerebella indicates that Cacna1atg - la mice have progressive PC and granule cell loss upon aging . Scale bar , 200 μm . . B , D . Calbindin ( green ) and 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ) ( blue ) staining of micecerebellum indicates that there are swollen PC axons ( arrows ) in the granular layers of the Cacna1atg - lamicecerebellum at day 25 ( B ) and the Cacna2d2du - 2Jmice at day 50 ( D ) . Scale bar , 20 μmC , E . The TEM of the axons at the granular layers of the cerebellum showing that many PC axons in Cacna1atg - la and Cacna2d2du - 2J mice are swollen and accumulated with numerous mitochondria , autophagosome / MVBs - like structures ( red arrows ) , and various cytoplasmic vesicles or aberrant membrane structures , including stacks of cisternal membranes ( blue arrows ) , and folded ER ( yellow arrows ) . Scale bars for right two panels are 2 μm . Other scale bars are 500 nmF , G . LC3II and p62 are slightly increased in 15 - days - old Cacna1atg - la mice and 50 - days - old Cacna2d2du - 2J mice . b , d are quantification of a and c . Data are presented as means ± SEM . ( * : p < 0 . 05 ) .

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Fig 4A. Calbindin and hematoxylin staining of mice cerebella indicates that Cacna1atg-la mice have progressive PC and granule cell loss upon aging. Scale bar, 200 μm.. B, D. Calbindin (green) and 4',6-diamidino-2-phenylindole (DAPI)(blue) staining of micecerebellum indicates that there are swollen PC axons (arrows) in the granular layers of the Cacna1atg-lamicecerebellum at day 25 (B) and the Cacna2d2du-2Jmice at day 50 (D). Scale bar, 20 μmC, E. The TEM of the axons at the granular layers of the cerebellum showing that many PC axons in Cacna1atg-la and Cacna2d2du-2J mice are swollen and accumulated with numerous mitochondria, autophagosome/MVBs-like structures (red arrows), and various cytoplasmic vesicles or aberrant membrane structures, including stacks of cisternal membranes (blue arrows), and folded ER (yellow arrows). Scale bars for right two panels are 2 μm. Other scale bars are 500 nmF, G. LC3II and p62 are slightly increased in 15-days-old Cacna1atg-la mice and 50-days-old Cacna2d2du-2J mice. b, d are quantification of a and c. Data are presented as means ± SEM. (*: p < 0.05).


Fig 5A . CACNA1A co - localizes with LAMP1 in primary cerebellar cultures of both CTL and Cacna1atg - la mutants . Scale bars , 20 μm . B . CACNA1A is present as punctae on the Vacuolin - 1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg - la mutants . Scale bars , 20 μm . C . Quantification of LAMP1 positive organelles that are also CACNA1A positive . Data are presented as means ± SD . ( ns : not significant ) . D . CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient .

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Fig 5A. CACNA1A co-localizes with LAMP1 in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm.B. CACNA1A is present as punctae on the Vacuolin-1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm.C. Quantification of LAMP1 positive organelles that are also CACNA1A positive. Data are presented as means ± SD. (ns: not significant).D. CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient.


Fig 6A . LysoTracker Red staining of the primary cerebella neurons from Cacna1atg - la mice and WT controls . The control cells show big bright punctae , while the mutant cells have much dimmer and smaller punctae . B , C and D . Quantification of LysoTracker relative intensity ( " n " represents the counted cell number ) , LysoTracker positive punctae number and size per cell ( " n " represents the counted image number ) . E . Lysosomal marker LAMP1 co - localizes with autophagosome marker LC3 in CTL but not Cacna1atg - la mutant cells . Scale bar , 20 μm . F . Quantification of LC3 and LAMP1 co - localization . Data are presented as means ± SD . ( ns : not significant ; * * * : p < 0 . 001 ) .

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Fig 6A. LysoTracker Red staining of the primary cerebella neurons from Cacna1atg-la mice and WT controls. The control cells show big bright punctae, while the mutant cells have much dimmer and smaller punctae. B, C and D. Quantification of LysoTracker relative intensity ("n" represents the counted cell number), LysoTracker positive punctae number and size per cell ("n" represents the counted image number). E. Lysosomal marker LAMP1 co-localizes with autophagosome marker LC3 in CTL but not Cacna1atg-la mutant cells. Scale bar, 20 μm. F. Quantification of LC3 and LAMP1 co-localization. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001).


Fig 7A . DQ - BSA labeled late endosomes fuse with LAMP1 labeled lysosomes in CTL but not the mutant cells . Scale bar , 20 μm . B . Quantification of DQ - BSA and LAMP1 co - localization in CTL and Cacna1atg - la cells . Data are presented as means ± SD . ( ns : not significant ; * * * : p < 0 . 001 ) C . Bepridil but not ω - agatoxin TK ( ω - Aga TK ) treatment reduces lysosomal fusion in cultured cerebellar cells . DQ - BSA ( Green ) labeled late endosomes co - localize with LAMP1 ( Red ) labeled lysosomes with / without ω - Aga TK ( 1 µM ) or Bepridil ( 10 µM ) treatment . Scale bar , 20 μm . D . Quantification of DQ - BSA and LAMP1 co - localization with ω - Aga TK or Bepridil treatment . Data are presented as means ± SD . ( ns : not significant ; * * : p < 0 . 001 ) .

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Fig 7A. DQ-BSA labeled late endosomes fuse with LAMP1 labeled lysosomes in CTL but not the mutant cells. Scale bar, 20 μm. B. Quantification of DQ-BSA and LAMP1 co-localization in CTL and Cacna1atg-la cells. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001) C. Bepridil but not ω-agatoxin TK (ω-Aga TK) treatment reduces lysosomal fusion in cultured cerebellar cells. DQ-BSA (Green) labeled late endosomes co-localize with LAMP1 (Red) labeled lysosomes with/without ω-Aga TK (1 µM) or Bepridil (10 µM) treatment. Scale bar, 20 μm. D. Quantification of DQ-BSA and LAMP1 co-localization with ω-Aga TK or Bepridil treatment. Data are presented as means ± SD. (ns: not significant; **: p < 0.001).


Figure 1 ( A ) Immunoblot analysis of caspase - 1 and gasdermin D in WT or Nlrp3 - / - unprimed and LPS - primed BMDMs left untreated [ Medium alone ( Med . ) ] or assessed 20 hr after stimulation with the supernatant ( Sup . ) of or assessed 20 hr after infected with WT C . perfringens ( WT C . per ) , ΔpfoA C . perfringens ( ΔpfoA ) , Δcpa C . perfringens ( Δcpa , lacking lecithinase ) or ΔpfoAΔcpa C . perfringens ( ΔpfoAΔcpa ) at MOI 100 . ( C ) Immunoblot analysis of caspase - 1 , gasdermin D and GAPDH ( loading control ) in WT or mutant BMDMs left untreated or LPS - primed and assessed 3 hr after stimulation with lecithinase ( Lec . ) . ( D ) Release of IL - 1β ( top ) , IL - 18 ( middle - top ) , death ( middle - bottom ) and TNF ( bottom ) of BMDMs after treatment as in C .

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Figure 1(A) Immunoblot analysis of caspase-1 and gasdermin D in WT or Nlrp3-/- unprimed and LPS-primed BMDMs left untreated [Medium alone (Med.)] or assessed 20 hr after stimulation with the supernatant (Sup.) of or assessed 20 hr after infected with WT C. perfringens (WT C. per), ΔpfoA C. perfringens (ΔpfoA), Δcpa C. perfringens (Δcpa, lacking lecithinase) or ΔpfoAΔcpa C. perfringens (ΔpfoAΔcpa) at MOI 100.(C) Immunoblot analysis of caspase-1, gasdermin D and GAPDH (loading control) in WT or mutant BMDMs left untreated or LPS-primed and assessed 3 hr after stimulation with lecithinase (Lec.).(D) Release of IL-1β (top), IL-18 (middle-top), death (middle-bottom) and TNF (bottom) of BMDMs after treatment as in C.


Figure 2 ( A ) Concentration of IL - 18 in the serum of WT mice , 8 hr after intraperitoneal infection with 4 × 108 colony - forming units ( CFUs ) of C . perfringens ( WT C . per , n = 14 ) , ΔpfoA C . perfringens ( ΔpfoA , n = 13 ) , Δcpa C . perfringens ( Δcpa , n = 11 ) or ΔpfoAΔcpa C . perfringens ( ΔpfoAΔcpa , n = 12 ) . ( B ) Concentration of IL - 18 in the peritoneal fluid of WT mice , 4 hr after intraperitoneal infection with 4 × 108 CFUs of C . perfringens ( WT C . per , n = 14 ) , ΔpfoA C . perfringens ( ΔpfoA , n = 15 ) , Δcpa C . perfringens ( Δcpa , n = 17 ) or ΔpfoAΔcpa C . perfringens ( ΔpfoAΔcpa , n = 14 ) . ( C ) Concentration of IL - 18 in the serum of WT mice ( n = 10 ) , Nlrp3 - / - mice ( n = 9 ) , and Casp1 / 11 - / - mice ( n = 19 ) , 6 hr after intraperitoneal infection with 1 × 108 CFUs of C . perfringens . ( D ) Concentration of IL - 18 in the peritoneal fluid of WT mice ( n = 10 ) , Nlrp3 - / - mice ( n = 9 ) , and Casp1 / 11 - / - mice ( n = 19 ) , 3 hr after intraperitoneal infection as described in C . ( G ) Survival of WT ( n = 19 ) , Nlrp3 - / - ( n = 26 ) and Casp1 - / - ( n = 10 ) mice , after intraperitoneal administration of 0 . 312 Units / ml of lecithinase ( Lec . ) . ( J ) Survival of WT mice administered with either PBS ( n = 20 ) or MCC950 ( n = 18 ) , followed by intraperitoneal administration of 0 . 312 Units / ml of lecithinase ( Lec . ) as in H .

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

Figure 2(A) Concentration of IL-18 in the serum of WT mice, 8 hr after intraperitoneal infection with 4 × 108 colony-forming units (CFUs) of C. perfringens (WT C. per, n = 14), ΔpfoA C. perfringens (ΔpfoA, n = 13), Δcpa C. perfringens (Δcpa, n = 11) or ΔpfoAΔcpa C. perfringens (ΔpfoAΔcpa, n = 12). (B) Concentration of IL-18 in the peritoneal fluid of WT mice, 4 hr after intraperitoneal infection with 4 × 108 CFUs of C. perfringens (WT C. per, n = 14), ΔpfoA C. perfringens (ΔpfoA, n = 15), Δcpa C. perfringens (Δcpa, n = 17) or ΔpfoAΔcpa C. perfringens (ΔpfoAΔcpa, n = 14). (C) Concentration of IL-18 in the serum of WT mice (n = 10), Nlrp3-/- mice (n = 9), and Casp1/11-/- mice (n = 19), 6 hr after intraperitoneal infection with 1 × 108 CFUs of C. perfringens. (D) Concentration of IL-18 in the peritoneal fluid of WT mice (n = 10), Nlrp3-/- mice (n = 9), and Casp1/11-/- mice (n = 19), 3 hr after intraperitoneal infection as described in C.(G) Survival of WT (n = 19), Nlrp3-/- (n = 26) and Casp1-/- (n = 10) mice, after intraperitoneal administration of 0.312 Units/ml of lecithinase (Lec.).(J) Survival of WT mice administered with either PBS (n = 20) or MCC950 (n = 18), followed by intraperitoneal administration of 0.312 Units/ml of lecithinase (Lec.) as in H.


Figure 3 ( A ) Confocal microscopy analysis of CD11b ( green ) and lecithinase ( red ) in unprimed WT BMDMs left untreated [ Medium alone ( Med . ) ] or assessed 1 hr after stimulation with Alexa Fluor 568 labelled lecithinase ( AF568 - Lec . ) in the absence or presence of cytochalasin D ( Cyto D ; 50 μM ) or methyl - β - cyclodextrin ( MCD ; 5 mM ) . ( D ) Immunoblot analysis of caspase - 1 and GSDMD of unprimed or LPS - primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase ( Lec . ) , or 3 hr after stimulation with perfringolysin O ( PFO ) , or 30 min after stimulation with nigericin ( Nig . ) in the absence or presence of methyl - β - cyclodextrin ( MCD ; 5 mM ) . ( E ) Release of IL - 1β ( left ) and IL - 18 ( middle ) , and death ( right ) of WT BMDMs after treatment as in D . ( F ) Immunoblot analysis of caspase - 1 and GSDMD of unprimed or LPS - primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase ( Lec . ) , or 4 hr after infection of with S . Typhimurium ( S . Tm . ; MOI , 5 ) , or 30 min after stimulation with nigericin in the absence or presence of cytochalasin D ( Cyto D ; 50 μM ) . ( G ) Release of IL - 1β ( left ) and IL - 18 ( middle ) , and death ( right ) of WT BMDMs after treatment as in F . ( H ) Immunoblot analysis of caspase - 1 and GSDMD of unprimed or LPS - primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase ( Lec . ) , or 4 hr after infection of with S . Typhimurium ( S . Tm . ; MOI , 5 ) , or 30 min after stimulation with nigericin in the absence or presence of latrunculin B ( Lat B ; 1 μg / ml ) . ( I ) Release of IL - 1β ( left ) and IL - 18 ( middle ) , and death ( right ) of WT BMDMs after treatment as in H .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy analysis of CD11b (green) and lecithinase (red) in unprimed WT BMDMs left untreated [Medium alone (Med.)] or assessed 1 hr after stimulation with Alexa Fluor 568 labelled lecithinase (AF568-Lec.) in the absence or presence of cytochalasin D (Cyto D; 50 μM) or methyl-β-cyclodextrin (MCD; 5 mM).(D) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 3 hr after stimulation with perfringolysin O (PFO), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of methyl-β-cyclodextrin (MCD; 5 mM). (E) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in D. (F) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 4 hr after infection of with S. Typhimurium (S. Tm.; MOI, 5), or 30 min after stimulation with nigericin in the absence or presence of cytochalasin D (Cyto D; 50 μM). (G) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in F. (H) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 4 hr after infection of with S. Typhimurium (S. Tm.; MOI, 5), or 30 min after stimulation with nigericin in the absence or presence of latrunculin B (Lat B; 1 μg/ml). (I) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in H.


Figure 4 ( B ) Confocal microscopy analysis of LAMP1 ( green ) and lecithinase ( red ) in WT BMDMs left untreated or assessed 1 hr after stimulation with AF568 - lecithinase . ( C ) Three - dimensional surface rendering of LAMP1 ( green ) and lecithinase ( red ) shown in B . ( D ) Line scan values for LAMP1 , AF568 - lecithinase , and DAPI taken from the white dashed line in the gold - colored inset in B . These values were plotted and fitted with a single Gaussian equation ( Arbitrary Unit ( AU ) ) .

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

Figure 4(B) Confocal microscopy analysis of LAMP1 (green) and lecithinase (red) in WT BMDMs left untreated or assessed 1 hr after stimulation with AF568-lecithinase. (C) Three-dimensional surface rendering of LAMP1 (green) and lecithinase (red) shown in B. (D) Line scan values for LAMP1, AF568-lecithinase, and DAPI taken from the white dashed line in the gold-colored inset in B. These values were plotted and fitted with a single Gaussian equation (Arbitrary Unit (AU)).


Figure 5 ( A ) Immunoblot analysis of caspase - 1 and GSDMD of unprimed or LPS - primed WT BMDMs left untreated [ Medium alone ( Med . ) ] or assessed 3 hr after stimulation with lecithinase ( Lec . ) , or 30 min after stimulation with nigericin ( Nig . ) in the absence or presence of bafilomycin A ( Baf A ; 100nm ) . ( C ) Immunoblot analysis of caspase - 1 and GSDMD of unprimed or LPS - primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase ( Lec . ) , or 16 hr after infection with F . novicida ( F . novi . , MOI 100 ) , or 30 min after stimulation with nigericin ( Nig . ) in the absence or presence of ammonium chloride ( NH4Cl ; 20 μM ) . ( B ) Release of IL - 1β ( left ) and IL - 18 ( middle ) , and death ( right ) of WT BMDMs after treatment as in A . ( D ) Release of IL - 1β ( left ) and IL - 18 ( middle ) , and death ( right ) of WT BMDMs after treatment as in C . ( F ) Flow cytometry analysis of WT BMDMs stained with acridine orange ( 1μg / ml ) and pre - treated with various inhibitors ( MCD , Cytochalasin D , Bafilomycin A , ammonium chloride ) for 30 min , and assessed 60 min after stimulation with lecithinase ( Lec . ) . WT BMDMs stained with acridine orange were also assessed 60 min after stimulation with heat - treated lecithinase ( Heat ) .

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

Figure 5(A) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated [Medium alone (Med.)] or assessed 3 hr after stimulation with lecithinase (Lec.), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of bafilomycin A (Baf A; 100nm). (C) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 16 hr after infection with F. novicida (F. novi., MOI 100), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of ammonium chloride (NH4Cl; 20 μM).(B) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in A. (D) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in C.(F) Flow cytometry analysis of WT BMDMs stained with acridine orange (1μg/ml) and pre-treated with various inhibitors (MCD, Cytochalasin D, Bafilomycin A, ammonium chloride) for 30 min, and assessed 60 min after stimulation with lecithinase (Lec.). WT BMDMs stained with acridine orange were also assessed 60 min after stimulation with heat-treated lecithinase (Heat).


Figure 6 ( E and F ) IncuCyte live - imaging analysis of the viability of BMDMs after treatment as in A . ( G and H ) IncuCyte live - imaging analysis of the viability of BMDMs after treatment as in C . ( I ) Release of IL - 1β ( top ) , IL - 18 ( middle ) and death ( bottom ) of WT or Ninj1 - / - BMDMs left untreated or LPS - primed and assessed for a range of time points after stimulation with lecithinase ( Lec . ) or 16 hr after transfection with LPS ( LPS trans . ) . ( J ) Immunoblot analysis of caspase - 1 , gasdermin D and NINJ1 of WT or Ninj1 - / - BMDMs left untreated or LPS - primed and assessed 3 hr after stimulation with lecithinase ( Lec . ) or 16 hr after transfection with LPS ( LPS trans . ) .

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

Figure 6(E and F) IncuCyte live-imaging analysis of the viability of BMDMs after treatment as in A.(G and H) IncuCyte live-imaging analysis of the viability of BMDMs after treatment as in C.(I) Release of IL-1β (top), IL-18 (middle) and death (bottom) of WT or Ninj1-/- BMDMs left untreated or LPS-primed and assessed for a range of time points after stimulation with lecithinase (Lec.) or 16 hr after transfection with LPS (LPS trans.).(J) Immunoblot analysis of caspase-1, gasdermin D and NINJ1 of WT or Ninj1-/- BMDMs left untreated or LPS-primed and assessed 3 hr after stimulation with lecithinase (Lec.) or 16 hr after transfection with LPS (LPS trans.).


Figure 1 ( C - C ' ' ' ) Terminal cell expressing reporters for membrane ( C ) , ER ( C ' ) and Golgi apparatus ( C ' ' ) . Red arrowheads : CD4 : : mIFP vesicles and associated markers at the tip of the cell . ( D ) Mean and 95 % confidence interval of tube and branch length during the first hour of growth . Numbers represent the rate of growth estimated from the slope of the curve + / - SD , p = 0 . 39 , Mann - Whitney test , n = 6 . ( E ) Distance of the tip of the tube , and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell . Line and shading represent mean and 95 % confidence interval , n = 4 . ( F - I ) Terminal cells expressing the general membrane marker CD4 : : mIFP in combination with different membrane or polarity markers : PH : : mCherry , a PIP2 sensor commonly used as apical plasma membrane marker ( F ' ) , an endogenously GFP - tagged Crb ( G ' ) , polarity proteins Par3 : : YFP ( H ' ) and Par6 : : mCherry ( H ' ' ) . The boxed region in ( H ' ' ' ) is shown at higher magnification in ( I ) over 4 time points . Red arrowheads : CD4 : : mIFP vesicles and associated markers at the tip of the cell .

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

Figure 1(C-C''') Terminal cell expressing reporters for membrane (C), ER (C') and Golgi apparatus (C''). Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.(D) Mean and 95% confidence interval of tube and branch length during the first hour of growth. Numbers represent the rate of growth estimated from the slope of the curve +/-SD, p=0.39, Mann-Whitney test, n=6.(E) Distance of the tip of the tube, and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell. Line and shading represent mean and 95% confidence interval, n=4.(F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points. Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.


Figure 2TEM tomograms from high - pressure frozen embryos . ( A - A ' ) Low magnification ( 2 . 5 nm voxels ) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell . ( A ' ' - A ' ' ' ) 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell . ( B ) Example of an individual high - resolution ( 0 . 78nm voxels ) tomograms used to generate 3D reconstructions . ( C ) Low resolution tomogram of another cell ; the dotted box is magnified in ( D ) . ( D ) High - resolution tomogram . ( D ' ) Model derived from 4 serial 3D reconstructions including the one shown in ( D ) .

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

Figure 2TEM tomograms from high-pressure frozen embryos. (A-A') Low magnification (2.5 nm voxels) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell. (A''-A''') 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell.  (B) Example of an individual high-resolution (0.78nm voxels) tomograms used to generate 3D reconstructions.(C) Low resolution tomogram of another cell; the dotted box is magnified in (D). (D) High-resolution tomogram. (D') Model derived from 4 serial 3D reconstructions including the one shown in (D).


Figure 3 ( B ) Distribution of three commonly seen classes of vesicles , expressed as their density per area of cytoplasm screened , in the two regions of the cell . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . n = 62 tomograms from two different cells . ( C ) Representative examples for each vesicle class are shown next to the quantification plots . ( D ) Distribution of endocytic events at the apical or basal membrane compartment , expressed as density per membrane surface screened . Left shows tomograms corresponding to the cell body and branch , and right shows tomograms corresponding to the tip of the cell . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . n = 48 tomograms from two different cells .

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

Figure 3(B) Distribution of three commonly seen classes of vesicles, expressed as their density per area of cytoplasm screened, in the two regions of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=62 tomograms from two different cells. (C) Representative examples for each vesicle class are shown next to the quantification plots. (D) Distribution of endocytic events at the apical or basal membrane compartment, expressed as density per membrane surface screened. Left shows tomograms corresponding to the cell body and branch, and right shows tomograms corresponding to the tip of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=48 tomograms from two different cells.


Figure 4Distribution of the plasma membrane reporter PH : : GFP in control cells ( A - A ' ' ) and in cells where dynamin activity had been blocked using a temperature - sensitive allele of dynamin ( shibirets In ( B ) , was inactivated at the onset of tube formation ( C - D ) Distribution of PH : : GFP fluorescence intensity in control and shibirets cells . Data from 1 - 2 minute - interval time lapses was collected in windows of 20 minutes each ( except for t = 0 ) . ( C ) Proportion of signal in the apical and in the basal membrane compartment over time in control cells ( n = 4 ) and also in cells where dynamin was inactivated ( n = 5 ) , * p = 0 . 015 , Mann - Whitney test . ( D ) Total fluorescence intensity of PH : : GFP over time in controls ( n = 4 ) and in cells where dynamin was inactivated ( n = 5 ) , n . s . : p = 0 . 063 , Mann - Whitney test . Distribution of the plasma membrane reporter PH : : GFP in cells where dynamin activity had been blocked using a temperature - sensitive allele of dynamin ( shibirets in ( E ) it was inactivated ~ 15 minutes after the tube had begun to form . ( F - G ) Distribution of the luminal reporter Gasp : : GFP in control cells ( F ) and in cells where dynamin was inactivated ( G ) . Arrowheads point to protrusions sprouting from the lumen of the terminal cell . ( H - I ) Distribution of the general plasma membrane marker CD4 : : mIFP in combination with the Clathrin light chain fused to GFP ( CLC : : GFP , H ) and with Dynamin : : YFP ( I ) .

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 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

Figure 4Distribution of the plasma membrane reporter PH::GFP in control cells (A-A'') and in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets In (B), was inactivated at the onset of tube formation (C-D) Distribution of PH::GFP fluorescence intensity in control and shibirets cells. Data from 1-2 minute-interval time lapses was collected in windows of 20 minutes each (except for t=0). (C) Proportion of signal in the apical and in the basal membrane compartment over time in control cells (n=4) and also in cells where dynamin was inactivated (n=5), *p=0.015, Mann-Whitney test. (D) Total fluorescence intensity of PH::GFP over time in controls (n=4) and in cells where dynamin was inactivated (n=5), n.s.: p=0.063, Mann-Whitney test. Distribution of the plasma membrane reporter PH::GFP in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets in (E) it was inactivated ~15 minutes after the tube had begun to form. (F-G) Distribution of the luminal reporter Gasp::GFP in control cells (F) and in cells where dynamin was inactivated (G). Arrowheads point to protrusions sprouting from the lumen of the terminal cell. (H-I) Distribution of the general plasma membrane marker CD4::mIFP in combination with the Clathrin light chain fused to GFP (CLC::GFP, H) and with Dynamin::YFP (I).


Figure 5 ( A - F ) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos . ( A ) Control . ( B - F ) Kept at 34ºC for 1 ( B , E ) or for 2 hours ( C , D , F ) . Boxed regions in ( A ) and ( B ) are magnified in ( A ' ' ) and ( B ' ' ) . ( A ' , B ' ) Reconstructed 3D models . The tube membrane ( cyan ) is characterized by the electron - dense apical extracellular matrix ( aECM ) . Invaginations with connections that can be traced to the tube membrane are red , invaginations from the basal plasma membrane are green . The basal membrane is light pink . ( C - D ' ) shibirets terminal cell after 2 hours at 34ºC . ( C ' ) 3D reconstruction , color coding for basal and tube membrane is the same as above . Membrane that is continuous with both the apical and the basal membrane is shown in green , purple and dark blue . ( D - D ' ) higher magnification of a region in the same cell at a level where membrane sheets ( purple , dark blue ) bridge the apical and basal plasma membrane domains . ( E - E ' ' ) Two focal planes and model of a tomogram from the cell shown in ( B ) where a basal ( pink arrowhead ) and an apical invagination ( blue ) are seen in close proximity ( closest distance is marked by cyan and blue arrowheads ) . ( F - F ' ) apical membrane overgrowth regions of the cell shown in ( C ) . The genotype of the embryos was shibirets ; btl > KDEL : : RFP , Par3 : : YFP . The cell shown in ( C - D ) was found and acquired without the CLEM approach .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos. (A) Control. (B-F) Kept at 34ºC for 1 (B, E) or for 2 hours (C, D, F). Boxed regions in (A) and (B) are magnified in (A'') and (B''). (A', B') Reconstructed 3D models. The tube membrane (cyan) is characterized by the electron-dense apical extracellular matrix (aECM). Invaginations with connections that can be traced to the tube membrane are red, invaginations from the basal plasma membrane are green. The basal membrane is light pink. (C-D') shibirets terminal cell after 2 hours at 34ºC. (C') 3D reconstruction, color coding for basal and tube membrane is the same as above. Membrane that is continuous with both the apical and the basal membrane is shown in green, purple and dark blue. (D-D') higher magnification of a region in the same cell at a level where membrane sheets (purple, dark blue) bridge the apical and basal plasma membrane domains. (E-E'') Two focal planes and model of a tomogram from the cell shown in (B) where a basal (pink arrowhead) and an apical invagination (blue) are seen in close proximity (closest distance is marked by cyan and blue arrowheads). (F-F') apical membrane overgrowth regions of the cell shown in (C). The genotype of the embryos was shibirets; btl>KDEL::RFP, Par3::YFP. The cell shown in (C-D) was found and acquired without the CLEM approach.


Figure 6 ( A - B ) Terminal cells expressing the membrane marker CD4 : : mIFP under btl - gal4 and FGFR : : GFP under its own promoter ( from the fTRG library ) . ( A ) Time lapse imaging of a control cell . Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4 : : mIFP and FGFR : : GFP . ( B ) shibirets cell imaged before dynamin inactivation , after 23 and 45 minutes of inactivation , and after 30 minutes of recovery . White arrowheads point to FGFR : : GFP accumulation at the apical compartment . ( C - E ) Single confocal planes of terminal cells expressing PH : : mCherry and stained for βPS - Integrin . The outline of the cells was traced using the PH : : mCherry signal and is shown as a blue dashed line . ( C ) Control . Blue arrowheads : βPS - Integrin signal in filopodia . ( D ) 1 hour at 34ºC , ( E ) 2 hours at 34ºC . ( C ' ' - E ' ' ) Orthogonal views of the lines shown in white in ( C - E ) . ( F - G ) Quantification of fluorescence intensity from stained embryos of FGFR : : GFP ( F ) and of βPS - Integrin ( G ) . Data are plotted as mean + / - SD ; significance was assessed using one - way ANOVA with Dunnett ' s correction for multiple comparisons . * * p = 0 . 0022 , * * * p = 0 . 0003 , * * * * p < 0 . 0001 . Number of cells analyzed , for ( F ) : Control n = 8 , 1hr at 34ºC n = 6 , Recovery n = 8 . ( G ) . For ( G ) : Control = 7 , 1hr at 34ºC = 8 , 2hrs at 34ºC = 8 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(A-B) Terminal cells expressing the membrane marker CD4::mIFP under btl-gal4 and FGFR::GFP under its own promoter (from the fTRG library). (A) Time lapse imaging of a control cell. Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4::mIFP and FGFR::GFP. (B) shibirets cell imaged before dynamin inactivation, after 23 and 45 minutes of inactivation, and after 30 minutes of recovery. White arrowheads point to FGFR::GFP accumulation at the apical compartment.(C-E) Single confocal planes of terminal cells expressing PH::mCherry and stained for βPS-Integrin. The outline of the cells was traced using the PH::mCherry signal and is shown as a blue dashed line. (C) Control. Blue arrowheads: βPS-Integrin signal in filopodia. (D) 1 hour at 34ºC, (E) 2 hours at 34ºC. (C''-E'') Orthogonal views of the lines shown in white in (C-E).(F-G) Quantification of fluorescence intensity from stained embryos of FGFR::GFP (F) and of βPS-Integrin (G). Data are plotted as mean +/- SD; significance was assessed using one-way ANOVA with Dunnett's correction for multiple comparisons. **p=0.0022, ***p=0.0003, ****p<0.0001. Number of cells analyzed, for (F): Control n=8, 1hr at 34ºC n=6, Recovery n=8. (G). For (G): Control= 7, 1hr at 34ºC= 8, 2hrs at 34ºC= 8.


Figure 7High time resolution imaging of the membrane marker CD4 : : mIFP expressed under btl - gal4 . ( A ) Example of a large CD4 vesicle ( arrowhead ) rapidly moving towards the tip of the terminal cell . ( B - D ) Analysis of 28 CD4 vesicles in five terminal cells . ( B ) Trajectories of CD4 vesicles with their original position mapped to the origin of the plot . ( C ) Rose diagram of the trajectories shown in ( B ) . ( D ) Speed of CD4 vesicles compared to speed of tube growth measured over 25 min . Data are plotted as mean + / - SD . ( E - H ) Terminal cells expressing the membrane reporter CD4 : : mIFP together with markers for vesicles of the endocytic pathway . ( E ) Rab5 : : YFP , red arrowheads point to a Rab5 - positive vesicle ; the white arrowhead shows the same vesicle once it lost the Rab5 signal . ( F ) FYVE : : GFP , a PI3P reporter and Spin : : mRFP , a lysosomal marker . Black arrowheads point to small FYVE : : GFP vesicles and the squared area shows large ones at the tip . ( G ) Par6 : : mCherry and endogenously labelled Rab7 : : YFP . ( H ) Percentage of CD4 : : mIFP vesicles that carry the indicated markers . Data are plotted as mean + / - SD . For each marker we analyzed at least 20 minutes of cell growth with 10 - 20 timepoints . Number of cells analyzed : Par3 , n = 2 ; Rab5 , n = 3 ; FYVE : : GFP , n = 4 ; FYVE : : GFP - Spin : : RFP ( which includes single - and double - positive vesicles ) , n = 3 ; dLamp1 , n = 2 ; Rab7 , n = 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7High time resolution imaging of the membrane marker CD4::mIFP expressed under btl-gal4. (A) Example of a large CD4 vesicle (arrowhead) rapidly moving towards the tip of the terminal cell. (B-D) Analysis of 28 CD4 vesicles in five terminal cells.(B) Trajectories of CD4 vesicles with their original position mapped to the origin of the plot. (C) Rose diagram of the trajectories shown in (B). (D) Speed of CD4 vesicles compared to speed of tube growth measured over 25 min. Data are plotted as mean +/-SD.(E-H) Terminal cells expressing the membrane reporter CD4::mIFP together with markers for vesicles of the endocytic pathway. (E) Rab5::YFP, red arrowheads point to a Rab5-positive vesicle; the white arrowhead shows the same vesicle once it lost the Rab5 signal. (F) FYVE::GFP, a PI3P reporter and Spin::mRFP , a lysosomal marker. Black arrowheads point to small FYVE::GFP vesicles and the squared area shows large ones at the tip. (G) Par6::mCherry and endogenously labelled Rab7::YFP. (H) Percentage of CD4::mIFP vesicles that carry the indicated markers. Data are plotted as mean +/-SD. For each marker we analyzed at least 20 minutes of cell growth with 10-20 timepoints. Number of cells analyzed: Par3, n=2; Rab5, n=3; FYVE::GFP, n=4; FYVE::GFP-Spin::RFP (which includes single- and double-positive vesicles), n=3; dLamp1, n=2; Rab7, n=5.


Figure 8 ( A - F ) Terminal cells expressing CD4 : : mIFP and FYVE : : GFP under btl - gal4 . The outline of the cells was traced using the CD4 : : mIFP signal and is shown as a blue dashed line . ( A - C ) Control cell . ( D - F ) shibirets cell before 34ºC ( D ) , after 30 minutes at 34ºC ( E ) and after 30 minutes of recovery ( F ) . Red arrowheads : FYVE : : GFP puncta . ( G - H ) Number of FYVE : : GFP vesicles in 4 control cells ( G ) and in 6 shibirets cells ( H ) . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . Significance in ( H ) was assessed using one - way ANOVA with Geisser - Greenhouse correction for paired data and Dunnett ' s multiple comparisons test . * p = 0 . 0288 , * * * p = 0 . 0016 . ( I ) shrbO3 / G5 mutant cell expressing CD4 : : mIFP under btl - gal4 . Arrowhead : CD4 : : mIFP aggregation . ( J - K ) Terminal cells over - expressing Shrb : : GFP under btl - gal4 together with PH : : mCherry ( J ) , or together with Spin : : RFP and CD4 : : mIFP ( K ) . Arrowheads : Shrb : : GFP accumulations . ( L - M ) Dorsal trunk cells stained for Serp . In control embryos ( L ) , Serp is seen exclusively in the tracheal lumen . The cells themselves are not visible in these images . In embryos expressing Shrb : : GFP under btl - gal4 ( M ) , Serp is also seen inside the cells , usually in association with or surrounded by Shrb : : GFP . Boxed regions are magnified in ( L ' - M ' ) and shown as single confocal planes .

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

Figure 8(A-F) Terminal cells expressing CD4::mIFP and FYVE::GFP under btl-gal4. The outline of the cells was traced using the CD4::mIFP signal and is shown as a blue dashed line. (A-C) Control cell. (D-F) shibirets cell before 34ºC (D), after 30 minutes at 34ºC (E) and after 30 minutes of recovery (F). Red arrowheads: FYVE::GFP puncta. (G-H) Number of FYVE::GFP vesicles in 4 control cells (G) and in 6 shibirets cells (H). Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. Significance in (H) was assessed using one-way ANOVA with Geisser-Greenhouse correction for paired data and Dunnett's multiple comparisons test. *p=0.0288, ***p=0.0016. (I) shrbO3/G5 mutant cell expressing CD4::mIFP under btl-gal4. Arrowhead: CD4::mIFP aggregation.(J-K) Terminal cells over-expressing Shrb::GFP under btl-gal4 together with PH::mCherry (J), or together with Spin::RFP and CD4::mIFP (K). Arrowheads: Shrb::GFP accumulations.(L-M) Dorsal trunk cells stained for Serp. In control embryos (L), Serp is seen exclusively in the tracheal lumen. The cells themselves are not visible in these images. In embryos expressing Shrb::GFP under btl-gal4 (M), Serp is also seen inside the cells, usually in association with or surrounded by Shrb::GFP. Boxed regions are magnified in (L'-M') and shown as single confocal planes.


Figure 1Cortical malformations observed in six novel BBSOAS patients Patient ( P ) 1 shows abnormally elongated occipital convolutions ( arrowheads in A ) , whereas abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals ( P2 - P6 B ' , C ' and C ' ' highlight the abnormally convoluted regions boxed in B and C . Cortical malformations observed in six novel BBSOAS patients Patient ( P ) 1 shows abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals ( P2 - P6 ; ( H , H ' ) TBR2 ( green in H ) , SOX2 ( red in H ) and NR2F1 ( yellow in H ' ) immunofluorescence ( IF ) of a GW11 section of human neocortex Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; oSVZ : outer subventricular zone ; SVZ : subventricular zone ; VZ : ventricular zone . ( I ) NR2F1 expression ( quantified by pixel intensity ) at different L - M levels and in different regions along the A - P extent of the cortex , as indicated . n ≥ 3 sections from n = 1 fetal brain . Data information the number of positive cells was quantified in 100µm - width boxes , randomly placed across the cortex . In graphs , data are represented as means ± SEM . 2 - way ANOVA ( I ) ( * P < 0 . 05 , * * * P < 0 . 001 ) . ( J - L ' ) NR2F1 ( red ) IF of human GW14 neocortex , showing expression levels in the CP ( K , L ) and VZ ( K ' , L ' ) around primary convolutions in the posterior - most cortex High NR2F1 is detected in the neurogenic area of a gyrus ( L ' ) . Data information : Nuclei ( blue ) were stained with DAPI . In Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; oSVZ : outer subventricular zone ; SVZ : subventricular zone ; VZ : ventricular zone . ( M , M ' ) NR2F1 level as measured on single cells in the VZ ( M ) and CP ( M ' ) of sulci and gyri n ≥ 4 sections from n = 1 fetal brain . ( N - O ) NR2F1 ( red ) and TBR2 ( green ) immunostaining NR2F1 level as measured in single TBR2 + IPs by pixel intensity is shown in graph ( O ) . n ≥ 4 convolutions from n = 1 fetal brain ( n = 2 sections ) . Data information In graphs , data are represented as means ± SEM . Student t - test ( M , M ' , O ( * P < 0 . 05 , * * * P < 0 . 001 ) . ( P - R ) NR2F1 ( red ) and HOPX ( green ) immunostaining Virtually all HOPX + bRGs are also NR2F1 + ( magnification in P ' ' ) and their number is greatly increased in gyri ( P ' ' ) compared to sulci ( P ' ) , as quantified in ( R ) . At a single cell level , NR2F1 pixel intensity is higher in oSVZ HOPX + cells in gyri compared to sulci ( Q ) . n ≥ 4 sections from n = 1 fetal brain . Data information : Nuclei ( blue ) were stained with DAPI . the number of positive cells was quantified in 100µm - width boxes , randomly placed across the cortex . In graphs , data are represented as means ± SEM . Student t - test Q , R ) ( * P < 0 . 05 , * * * P < 0 . 001 ) . oSVZ : outer subventricular

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

Figure 1Cortical malformations observed in six novel BBSOAS patients Patient (P)1 shows abnormally elongated occipital convolutions (arrowheads in A), whereas abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6 B', C' and C'' highlight the abnormally convoluted regions boxed in B and C.Cortical malformations observed in six novel BBSOAS patients Patient (P)1 shows abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6;(H,H') TBR2 (green in H), SOX2 (red in H) and NR2F1 (yellow in H') immunofluorescence (IF) of a GW11 section of human neocortex Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.(I) NR2F1 expression (quantified by pixel intensity) at different L-M levels and in different regions along the A-P extent of the cortex, as indicated. n ≥ 3 sections from n = 1 fetal brain. Data information the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. 2-way ANOVA (I) (*P<0.05, ***P<0.001).(J-L') NR2F1 (red) IF of human GW14 neocortex, showing expression levels in the CP (K,L) and VZ (K',L') around primary convolutions in the posterior-most cortex High NR2F1 is detected in the neurogenic area of a gyrus (L'). Data information: Nuclei (blue) were stained with DAPI. In Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.(M,M') NR2F1 level as measured on single cells in the VZ (M) and CP (M') of sulci and gyri n ≥ 4 sections from n = 1 fetal brain. (N-O) NR2F1 (red) and TBR2 (green) immunostaining NR2F1 level as measured in single TBR2+ IPs by pixel intensity is shown in graph (O). n ≥ 4 convolutions from n = 1 fetal brain (n = 2 sections). Data information In graphs, data are represented as means ± SEM. Student t-test (M,M',O (*P<0.05, ***P<0.001).(P-R) NR2F1 (red) and HOPX (green) immunostaining Virtually all HOPX+ bRGs are also NR2F1+ (magnification in P'') and their number is greatly increased in gyri (P'') compared to sulci (P'), as quantified in (R). At a single cell level, NR2F1 pixel intensity is higher in oSVZ HOPX+ cells in gyri compared to sulci (Q). n ≥ 4 sections from n = 1 fetal brain. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. Student t-test Q,R) (*P<0.05, ***P<0.001). oSVZ: outer subventricular zone


Figure 2 ( A ) Representative images of wild - type ( WT ) and mutant ( KO ) neurospheres obtained from E15 . 5 neocortices and cultured in vitro . The culture passage ( " step " ) corresponds to 3 days of culture after dissociation and re - plating . KO aggregates are more numerous and larger in size . Data information : Scale bars : 50µm . ( B ) Graph showing the number of neurospheres per P100 cell plate at different steps , as indicated . Nr2f1KO neurospheres proliferate for a longer time ( up to 30 passages tested ) , whereas WT cells exhaust around step 9 . n ≥ 3 culture wells from n = 2 batches . Data information : data are represented as means ± SEM . 2 - way ANOVA ( B ) ( * * P < 0 . 01 , * * * P < 0 . 001 ) . ( C ) Panel of WT and KO isolated neurospheres grown from day 1 to day 10 . Blue and red lines represent the diameter of WT and KO neurospheres , respectively . Data information Scale bars : 50µm . ( D , E ) PH3 ( green ; dividing cells ) IF on cross - sections of day 4 neurospheres . White arrowheads point at mitotic figures , which are quantified in ( E ) as percentage of dividing cells over DAPI - stained nuclei ( blue ) . n ≥ 3 neurospheres from n = 2 batches . Data information : data are represented as means ± SEM . Student t - test ( E ) ( * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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Figure 2(A) Representative images of wild-type (WT) and mutant (KO) neurospheres obtained from E15.5 neocortices and cultured in vitro. The culture passage ("step") corresponds to 3 days of culture after dissociation and re-plating. KO aggregates are more numerous and larger in size. Data information: Scale bars: 50µm.(B) Graph showing the number of neurospheres per P100 cell plate at different steps, as indicated. Nr2f1KO neurospheres proliferate for a longer time (up to 30 passages tested), whereas WT cells exhaust around step 9. n ≥ 3 culture wells from n = 2 batches. Data information: data are represented as means ± SEM. 2-way ANOVA (B) (**P<0.01, ***P<0.001).(C) Panel of WT and KO isolated neurospheres grown from day 1 to day 10. Blue and red lines represent the diameter of WT and KO neurospheres, respectively. Data information Scale bars: 50µm.(D,E) PH3 (green; dividing cells) IF on cross-sections of day 4 neurospheres. White arrowheads point at mitotic figures, which are quantified in (E) as percentage of dividing cells over DAPI-stained nuclei (blue). n ≥ 3 neurospheres from n = 2 batches. Data information: data are represented as means ± SEM. Student t-test (E) (**P<0.01, ***P<0.001). Scale bars: 50µm.