( A ) Immunofluorescence staining with anti - α - Spectrin ( red ) , anti - Vasa ( blue ) and anti - GFP ( green ) on adult ovary . The scale bar is 10 μm . ( B ) Immunofluorescence staining on PGCs of larvae ovary . The scale bar is 20 μm . ( C ) Immunofluorescence staining on GSCs and early spermatogonia in adult testis . The scale bar is 20 μm .

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(A) Immunofluorescence staining with anti-α-Spectrin (red), anti-Vasa (blue) and anti-GFP (green) on adult ovary. The scale bar is 10 μm. (B) Immunofluorescence staining on PGCs of larvae ovary. The scale bar is 20 μm. (C) Immunofluorescence staining on GSCs and early spermatogonia in adult testis. The scale bar is 20 μm.


( D ) Immunofluorescence staining on PGCs of larvae testis tissue . The scale bar is 20 μm . ( E ) Immunofluorescence staining with anti - α - Spectrin ( red ) and anti - Vasa ( green ) on testis from wild type or eggpl [ 1 ] flies . The scale bar is 10 μm .

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(D) Immunofluorescence staining on PGCs of larvae testis tissue. The scale bar is 20 μm. (E) Immunofluorescence staining with anti-α-Spectrin (red) and anti-Vasa (green) on testis from wild type or eggpl[1] flies. The scale bar is 10 μm.


( A ) Immunofluorescence staining with anti - BrdU ( red ) . The scale bar is 10 μm .

(A) Immunofluorescence staining with anti-BrdU (red). The scale bar is 10 μm.


( B ) The oviposition of wild type , Nos - Gal4 > UAS - eggpl - RNAi and eggpl [ 1 ] line over 21 days . Error bars show mean ± SEM , 3 biological replicates ( n = 3 ) were performed , and there were 10 female and 5 male adult flies in each biological replicate .

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(B) The oviposition of wild type, Nos-Gal4>UAS-eggpl-RNAi and eggpl[1] line over 21 days. Error bars show mean ± SEM, 3 biological replicates (n=3) were performed, and there were 10 female and 5 male adult flies in each biological replicate.


( F ) Immunofluorescence staining with anti - GFP on germaria from eggpl : : GFP knock - in flies with or without feeding fresh yeast paste on day 2 - , 7 - , 14 - and 21 . The dotted line indicates the examined area . The scale bar is 10 μm . ( G ) Quantification of the GFP intensity mean in the eggpl : : GFP knock - in line . The central band indicates the median , the box length indicates the interquartile range , and the upper and whisker indicate the maximum and minimum value respectively . The 10 biological replicates ( n = 10 ) were performed , one - way ANOVA , * * * P < 0 . 001 . The dashed line indicates the examined area .

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(F) Immunofluorescence staining with anti-GFP on germaria from eggpl::GFP knock-in flies with or without feeding fresh yeast paste on day 2-, 7-, 14- and 21. The dotted line indicates the examined area. The scale bar is 10 μm. (G) Quantification of the GFP intensity mean in the eggpl::GFP knock-in line. The central band indicates the median, the box length indicates the interquartile range, and the upper and whisker indicate the maximum and minimum value respectively. The 10 biological replicates (n=10) were performed, one-way ANOVA, ***P<0.001. The dashed line indicates the examined area.


( H - I ) Images of ovaries or mature eggs from flies of the indicated genotypes , feeding conditions , and days post - eclosion showing comparisons of the overall ovary sizes ( F ) and the numbers of retained matured eggs ( G ) . The scar bar is 500 μm . ( J - K ) Quantitative analysis of the ovary size ( H ) and the number of retained mature eggs . ( I ) under different food conditions . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . The 3 biological replicates ( n = 3 ) were performed .

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(H-I) Images of ovaries or mature eggs from flies of the indicated genotypes, feeding conditions, and days post-eclosion showing comparisons of the overall ovary sizes (F) and the numbers of retained matured eggs (G). The scar bar is 500 μm. (J-K) Quantitative analysis of the ovary size (H) and the number of retained mature eggs. (I) under different food conditions. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, *P<0.05, **P<0.01, ***P<0.001. The 3 biological replicates (n=3) were performed.


( B ) Quantification of the GFP signal in germaria from eggpl : : GFP knock - in flies of the indicated genotypes under different conditions . The central band indicates the median , the box length indicates the interquartile range , and the upper and whisker indicate the maximum and minimum value respectively , one - way ANOVA , * * P < 0 . 01 , * * * P < 0 . 001 , n = 10 , biological replicates .

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(B) Quantification of the GFP signal in germaria from eggpl::GFP knock-in flies of the indicated genotypes under different conditions. The central band indicates the median, the box length indicates the interquartile range, and the upper and whisker indicate the maximum and minimum value respectively, one-way ANOVA, **P<0.01, ***P<0.001, n = 10, biological replicates.


( C ) Immunofluorescence staining with anti - GFP on germaria with or without feeding fresh yeast paste on 7th day . The dashed line indicates the examined area . The scale bar is 10 μm .

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(C) Immunofluorescence staining with anti-GFP on germaria with or without feeding fresh yeast paste on 7th day. The dashed line indicates the examined area. The scale bar is 10 μm.


A . Immunoblotting of c - MYC and ATF4 in melanoma cells treated with NT - siRNA ( - ) or siASNS ( # 1 or # 2 ) for 72 hr .

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A. Immunoblotting of c-MYC and ATF4 in melanoma cells treated with NT-siRNA (-) or siASNS (#1 or #2) for 72 hr.


Reverse transcription with quantitative PCR ( RT - qPCR ) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS ( # 1 or # 2 ) ( B )

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

Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2) (B)


C . Reverse transcription with quantitative PCR ( RT - qPCR ) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS , siATF4 , or both ( C ) .

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C. Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS, siATF4, or both (C).


D . Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS , siATF4 , or both .

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D. Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS, siATF4, or both.


E . Proliferation of melanoma cells over indicated times after treatment with siASNS , siATF4 , or both .

E. Proliferation of melanoma cells over indicated times after treatment with siASNS, siATF4, or both.


F . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS ( # 1 or # 2 ) .

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F. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2).


Immunoblotting of c - MYC in melanoma cells treated for 72 hr with siASNS , L - Asn , or both ( G )

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Immunoblotting of c-MYC in melanoma cells treated for 72 hr with siASNS, L-Asn, or both (G)


H . Immunoblotting of c - MYC in A375 cells treated as indicated for 72 hr ( H ) .

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H. Immunoblotting of c-MYC in A375 cells treated as indicated for 72 hr (H).


A . Immunoblotting of c - MYC in A375 cells treated with NT - siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints ( left ) . Plot showing relative protein levels of c - MYC ( y - axis ) as a function of time lapsed ( x - axis ) in the left panel ( right ) .

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A. Immunoblotting of c-MYC in A375 cells treated with NT-siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints (left). Plot showing relative protein levels of c-MYC (y-axis) as a function of time lapsed (x-axis) in the left panel (right).


Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with NT - siRNA ( - ) or siASNS ( # 1 or # 2 ) ( B and C )

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

Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with NT-siRNA (-) or siASNS (#1 or #2) (B and C)


Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS , PLX - 4032 , or both ( D ) .

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Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, PLX-4032, or both (D).


E . Immunoblotting of indicated proteins in A375 cells cultured in L - Asn and treated with L - A ' ase , PLX - 4032 , or both for 72 hr .

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E. Immunoblotting of indicated proteins in A375 cells cultured in L-Asn and treated with L-A'ase, PLX-4032, or both for 72 hr.


F . A375 cells were treated with siASNS , SCH772984 , or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3 - β .

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F. A375 cells were treated with siASNS, SCH772984, or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3-β.


G . A375 cells were treated with siASNS , HA - GSK3 - β ( S9A ) , or both for 72 hr followed by immunoblotting of indicated proteins .

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G. A375 cells were treated with siASNS, HA-GSK3-β (S9A), or both for 72 hr followed by immunoblotting of indicated proteins.


H . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

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H. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.


I . A375 cells were treated as indicated for 72 hr followed by immunoblotting of c - MYC .

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I. A375 cells were treated as indicated for 72 hr followed by immunoblotting of c-MYC.


J . Immunoblotting of c - MYC and HA proteins in A375 cells treated as indicated for 72 hr .

J. Immunoblotting of c-MYC and HA proteins in A375 cells treated as indicated for 72 hr.


A . Immunoblotting of indicated proteins in melanoma cells treated with siASNS , sicMYC ( # 1 or # 2 ) , or a combination of siASNS and sicMYC ( # 1 or # 2 ) for 72 hr .

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A. Immunoblotting of indicated proteins in melanoma cells treated with siASNS, sicMYC (#1 or #2), or a combination of siASNS and sicMYC (#1 or #2) for 72 hr.


B . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS , sicMYC , or both .

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B. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, sicMYC, or both.


C . Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS , sicMYC , PLX - 4032 , or a combination of siASNS and sicMYC , or siASNS and PLX - 4032 .

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C. Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, sicMYC, PLX-4032, or a combination of siASNS and sicMYC, or siASNS and PLX-4032.


D . Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

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D. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.


E . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

E. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, HA-GSK3-β (S9A), or both.


F . A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins .

F. A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins.


Proliferation of melanoma cells over indicated times after treatment with siASNS , sicMYC , or both ( G ) ,

Proliferation of melanoma cells over indicated times after treatment with siASNS, sicMYC, or both (G),


Proliferation of melanoma cells over indicated times after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both ( H ) .

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Proliferation of melanoma cells over indicated times after treatment with siASNS, HA-GSK3-β (S9A), or both (H).


A . Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS , siSLC1A5 , siSLC7A5 , or a combination of siASNS and siSLC1A5 , or siASNS and siSLC7A5 .

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A. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, siSLC1A5, siSLC7A5, or a combination of siASNS and siSLC1A5, or siASNS and siSLC7A5.


B . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , siSLC7A5 , or both .

B. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC7A5, or both.


C . Proliferation of melanoma cells over indicated times after treatment with siASNS , siSLC7A5 , or both .

C. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC7A5, or both.


D . GC - MS - based estimation of intracellular amino acid levels in A375 cells treated with siASNS , siATF4 , or both ( top left ) ; siASNS , sicMYC , or both ( top right ) ; siASNS , siSLC1A5 , or both ( bottom left ) ; or siASNS , siSLC7A5 , or both ( bottom right ) . The fold change is relative to NT - siRNA - treated A375 cells .

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D. GC-MS-based estimation of intracellular amino acid levels in A375 cells treated with siASNS, siATF4, or both (top left); siASNS, sicMYC, or both (top right); siASNS, siSLC1A5, or both (bottom left); or siASNS, siSLC7A5, or both (bottom right). The fold change is relative to NT-siRNA-treated A375 cells.


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment ( E ) ,

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment (E),


Immunoblotting of indicated proteins in A375 cells 72 hr after , treatment with siASNS , sicMYC , or

Immunoblotting of indicated proteins in A375 cells 72 hr after , treatment with siASNS, sicMYC, or both


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS , siSLC7A5 , or both ( G )

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Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS, siSLC7A5, or both (G)


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , BCH , or both ( H )

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Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, BCH, or both (H)


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , HA - GSK3 - β ( S9A ) , or both ( I )

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Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, HA-GSK3-β (S9A), or both (I)


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS or indicated combinations ( J )

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Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS or indicated combinations (J)


Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , siSLC3A2 , or both ( K ) .

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, siSLC3A2, or both (K).


L . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , siSLC3A2 , or both .

L. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC3A2, or both.


M . Proliferation of melanoma cells over indicated times after treatment with siASNS , siSLC3A2 , or both .

M. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC3A2, or both.


A . Confocal microscopy image of PD myotubes ( cl . 3LE8 ) infected for 72 h with adenovirus containing TFEB ( PD + Ad - TFEB 72 h ) shows a dramatic reduction in the number of large LAMP1 - positive lysosomes ( red ) compared to that in untreated ( PD ) or adenovirus ( PD + Ad - null ) - treated PD myotubes . WT myotubes are shown on the left panel . Nuclei are stained with Hoechst ( blue ) . TFEB was detected with anti - Flag antibody ( green ) . B . Distribution of lysosomal size differs significantly in Ad - null and Ad - TFEB PD myotubes ( p = 6 . 32 × 10 − 8 ; Kolmogorov - Smirnov test ) . Lysosomal size is expressed as number of pixels representing lysosomal area ( LAMP1 - positive structures ) . The median lysosomal size of Ad - TFEB infected myotubes ( m = 367 . 13 pixels , n = 703 , range 208 - 2659 ) was significantly lower than that of Ad - null infected myotubes ( m = 491 . 16 pixels , n = 1395 , range 200 - 2857 ; p = 6 . 5 × 10 − 12 ; Wilcoxon rank sum test ) .

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A. Confocal microscopy image of PD myotubes (cl. 3LE8) infected for 72 h with adenovirus containing TFEB (PD + Ad-TFEB 72 h) shows a dramatic reduction in the number of large LAMP1-positive lysosomes (red) compared to that in untreated (PD) or adenovirus (PD + Ad-null)-treated PD myotubes. WT myotubes are shown on the left panel. Nuclei are stained with Hoechst (blue). TFEB was detected with anti-Flag antibody (green).B. Distribution of lysosomal size differs significantly in Ad-null and Ad-TFEB PD myotubes (p = 6.32 × 10−8; Kolmogorov-Smirnov test). Lysosomal size is expressed as number of pixels representing lysosomal area (LAMP1-positive structures). The median lysosomal size of Ad-TFEB infected myotubes (m = 367.13 pixels, n = 703, range 208-2659) was significantly lower than that of Ad-null infected myotubes (m = 491.16 pixels, n = 1395, range 200-2857; p = 6.5 × 10−12; Wilcoxon rank sum test).


C . Confocal microscopy image of PD myotubes infected for 24 h with Ad - TFEB shows relocation of lysosomes to the plasma membrane ( top ) . Images showing LAMP1 staining ( red ) on plasma membrane in a PD myotube infected with Ad - TFEB ( bottom ; arrows ) but not in a non - infected cell ( middle ) . Non - permeabilized cells were incubated with anti - LAMP1 antibody at 4 ° C for 40 min , followed by fixation and staining with secondary antibody .

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C. Confocal microscopy image of PD myotubes infected for 24 h with Ad-TFEB shows relocation of lysosomes to the plasma membrane (top). Images showing LAMP1 staining (red) on plasma membrane in a PD myotube infected with Ad-TFEB (bottom; arrows) but not in a non-infected cell (middle). Non-permeabilized cells were incubated with anti-LAMP1 antibody at 4°C for 40 min, followed by fixation and staining with secondary antibody.


D . Confocal microscopy images of live non - infected PD myotubes ( left ) or PD myotubes infected for 72 h with Ad - TFEB ( right ) show a dramatic reduction in the amount of accumulated glycogen in TFEB - treated cells . The cells were incubated with the fluorescent glucose ( 2 - NBDG ; green ) , extensively washed , and analysed by confocal microscopy . Bar : 10 µm for all panels .

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D. Confocal microscopy images of live non-infected PD myotubes (left) or PD myotubes infected for 72 h with Ad-TFEB (right) show a dramatic reduction in the amount of accumulated glycogen in TFEB-treated cells. The cells were incubated with the fluorescent glucose (2-NBDG; green), extensively washed, and analysed by confocal microscopy. Bar: 10 µm for all panels.


Confocal microscopy of live unstained fibres from a GFP - LC3 : WT mouse ( WT ) and from untreated ( GAA − / − ) or ERT - treated ( GAA − / − ; + ERT ) GFP - LC3 : GAA − / − mice . Images show typical centrally located autophagic build - up with multiple clusters of LC3 - positive autophagosomes . Structures like these are never seen in WT muscle . Autophagic build - up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles ( not shown ) , but only in 60 - 70 % of FDBfibres . Labelled recombinant human GAA ( red ) was administered i . v . into 3 - 4 month - old GFP - LC3 : GAA − / − mice ( n = 5 ) at a dose of 100 mg / kg twice with a 24 h interval . Mice were sacrificed the next day , and live fibres ( shown for EDLmuscle ) were analysed by confocal microscopy . The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles . At least 500 fibres were analysed for each condition . Bar : 10 µm .

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Confocal microscopy of live unstained fibres from a GFP-LC3:WT mouse (WT) and from untreated (GAA−/−) or ERT-treated (GAA−/−; +ERT) GFP-LC3:GAA−/− mice. Images show typical centrally located autophagic build-up with multiple clusters of LC3-positive autophagosomes. Structures like these are never seen in WT muscle. Autophagic build-up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles (not shown), but only in 60-70% of FDBfibres. Labelled recombinant human GAA (red) was administered i.v. into 3-4 month-old GFP-LC3:GAA−/− mice (n = 5) at a dose of 100 mg/kg twice with a 24 h interval. Mice were sacrificed the next day, and live fibres (shown for EDLmuscle) were analysed by confocal microscopy. The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles. At least 500 fibres were analysed for each condition. Bar: 10 µm.


A . Immunostaining of non - infected cells ( day 13 in differentiation medium ) and cells infected with a mutant form of TFEB ( TFEBmt ) with LAMP1 ( red ) and Flag ( green ) . Ad - TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium . Ad - TFEBmt - infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size , similar to that seen with TFEB .

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A. Immunostaining of non-infected cells (day 13 in differentiation medium) and cells infected with a mutant form of TFEB (TFEBmt) with LAMP1 (red) and Flag (green). Ad-TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium. Ad-TFEBmt-infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size, similar to that seen with TFEB.


B . Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction ; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt - treated samples may reflect the differences in the glycosylation pattern .

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B. Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt-treated samples may reflect the differences in the glycosylation pattern.


C . Western blot of protein lysates from untreated , Ad - null , TFEB , and TFEBmt - treated myotubes with anti - caspase - 3 antibody . No activated ( cleaved ) products are detected in any condition . α - Tubulin was used as a loading control .

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C. Western blot of protein lysates from untreated, Ad-null, TFEB, and TFEBmt-treated myotubes with anti-caspase-3 antibody. No activated (cleaved) products are detected in any condition. α-Tubulin was used as a loading control.


D . TUNEL assay shows the presence of some apoptotic cells in Ad - TFEB - infected cultures , but not in control cultures infected with adenovirus alone ( n = 3 ) . Bar : 10 µm for all panels .

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D. TUNEL assay shows the presence of some apoptotic cells in Ad-TFEB-infected cultures, but not in control cultures infected with adenovirus alone (n = 3). Bar: 10 µm for all panels.


A . Confocal microscopy images of live fibres derived from 3 to 4 month - old GFP - LC3 : WT ( top left ) , untreated GFP - LC3 : GAA − / − ( bottom left ) or TFEB - treated GFP - LC3 : GAA − / − ( right ) mice . All fibres were transfected with mCherry - LAMP1 to visualize lysosomes ( red ) . The effects of TFEB are clearly visible - overall reduction in lysosomal size , appearance of normal size lysosomes ( similar to those in the WT ) , and lysosomal docking to the plasma membrane ( inset ) . Bar : 10 µm .

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A. Confocal microscopy images of live fibres derived from 3 to 4 month-old GFP-LC3:WT (top left), untreated GFP-LC3:GAA−/− (bottom left) or TFEB-treated GFP-LC3:GAA−/− (right) mice. All fibres were transfected with mCherry-LAMP1 to visualize lysosomes (red). The effects of TFEB are clearly visible - overall reduction in lysosomal size, appearance of normal size lysosomes (similar to those in the WT), and lysosomal docking to the plasma membrane (inset). Bar: 10 µm.


B . FDBmuscle of a GAA − / − mouse was transfected with both GFP - TFEB and mCherry - LAMP1 ( GAA − / − + GFP - TFEB ) . Images were taken before ( left ) and after 4 h ( right ) of time - lapse microscopy . Lysosomal clearance is visible in the TFEB - transfected fibre at both time points . Lysosomes appear to " exit " the fibre ( inset and arrow ) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation ( green nuclei ) . Bar : 10 µm . C . The mean maximum velocity of lysosomes ( top ) and the number of large ( > 3 . 5 µm ) lysosomes ( bottom ) in untreated and TFEB - treated PD fibres ( note : all data for Flag - and GFP - TFEB - treated fibres are pooled ) . Lysosomal velocities were calculated from time - lapse images using ImageJ software with the manual tracking plug - in . For each condition the trajectories of multiple lysosomes were followed ( n = 26 untreated ; n = 43 TFEB - treated ) and the three highest velocity measurements per lysosome were recorded . In TFEB - treated fibres , the maximum velocity of lysosomes was significantly increased ( p = 2 . 07 × 10 − 17 ) and the number of large lysosomes was significantly decreased ( p = 1 . 0 × 10 − 3 ) . Ten untreated and 24 TFEB - treated fibres were analysed for the size calculations . * indicates statistically significant differences ( p ≤ 0 . 001 ; Student ' s t - test ) . Error bars represent 95 % confidence intervals .

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B. FDBmuscle of a GAA−/− mouse was transfected with both GFP-TFEB and mCherry-LAMP1 (GAA−/− + GFP-TFEB). Images were taken before (left) and after 4 h (right) of time-lapse microscopy. Lysosomal clearance is visible in the TFEB-transfected fibre at both time points. Lysosomes appear to "exit" the fibre (inset and arrow) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation (green nuclei). Bar: 10 µm.C. The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated PD fibres (note: all data for Flag- and GFP-TFEB-treated fibres are pooled). Lysosomal velocities were calculated from time-lapse images using ImageJ software with the manual tracking plug-in. For each condition the trajectories of multiple lysosomes were followed (n = 26 untreated; n = 43 TFEB-treated) and the three highest velocity measurements per lysosome were recorded. In TFEB-treated fibres, the maximum velocity of lysosomes was significantly increased (p = 2.07 × 10−17) and the number of large lysosomes was significantly decreased (p = 1.0 × 10−3). Ten untreated and 24 TFEB-treated fibres were analysed for the size calculations. * indicates statistically significant differences (p ≤ 0.001; Student's t-test). Error bars represent 95% confidence intervals.


Confocal microscopy images of live fibres from GFP - LC3 : GAA − / − mice . Muscle was transfected with mCherry - LAMP1 only . The image ( a single frame from the time - lapse series presented in Movie 5 ) shows lysosomes ( red ) , LC3 - positive autophagosomes ( green ) and a number of autolysosomes ( yellow ) . Muscle was transfected with Flag - TFEB and mCherry - LAMP1 . Massive formation of autolysosomes is indicated by yellow structures ; the three lower panels provide a snapshot of the process of exocytosis . The structures at the plasma membrane are labelled with both LC3 and LAMP1 , indicating that they represent amphisomes ( a product of fusion between autophagic vesicles and late endosomes ) or autolysosomes ( a product of fusion between autophagic vesicles and lysosomes ) . Bar : 10 µm for all panels .

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Confocal microscopy images of live fibres from GFP-LC3:GAA−/− mice.Muscle was transfected with mCherry-LAMP1 only. The image (a single frame from the time-lapse series presented in Movie 5) shows lysosomes (red), LC3-positive autophagosomes (green) and a number of autolysosomes (yellow).Muscle was transfected with Flag-TFEB and mCherry-LAMP1. Massive formation of autolysosomes is indicated by yellow structures; the three lower panels provide a snapshot of the process of exocytosis. The structures at the plasma membrane are labelled with both LC3 and LAMP1, indicating that they represent amphisomes (a product of fusion between autophagic vesicles and late endosomes) or autolysosomes (a product of fusion between autophagic vesicles and lysosomes). Bar: 10 µm for all panels.


Confocal microscopy images of live fibres from muscle - specific autophagy - deficient GAA − / − mice ( Atg7 : GAA DKO ) . Muscle was transfected with mCherry - LAMP1 only . Muscle was transfected with GFP - TFEB and mCherry - LAMP1 ( Atg7 : GAA DKO + GFP - TFEB ) . The TFEB - transfected fibres show realignment of the lysosomes and membrane detachment ( most striking in top and bottom panels ) similar to those in TFEB - transfected fibres from PD mice ( see Fig 4B inset ) . Lysosomes can be seen in the space between the fibre and plasma membrane ( arrows ) . Bar : 10 µm for all panels . The mean maximum velocity of lysosomes ( top ) and the number of large ( > 3 . 5 µm ) lysosomes ( bottom ) in untreated and TFEB - treated autophagy - deficient Atg7 : GAA DKO fibres . The increase in maximum velocity ( 41 % ) is significant ( p = 2 . 729 × 10 − 18 ; n = 57 lysosomes for untreated ; n = 52 lysosomes for TFEB - treated ) , but there is only a slight trend toward smaller lysosomal size ( p = 7 . 0 × 10 − 2 ; n = 10 fibres for untreated ; n = 21 fibres for TFEB - treated ) . The corresponding values from GAA − / − mice are presented for comparison ( for TFEB - treated condition : n = 19 lysosomes for velocity measurements , and n = 10 fibres for the lysosomal size measurements ) . * indicates statistically significant differences ( p ≤ 1 . 0 × 10 − 5 for the top panel and p ≤ 0 . 01 for the lower panel ; Student ' s t - test ) .

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Confocal microscopy images of live fibres from muscle-specific autophagy-deficient GAA−/− mice (Atg7:GAA DKO).Muscle was transfected with mCherry-LAMP1 only.Muscle was transfected with GFP-TFEB and mCherry-LAMP1 (Atg7:GAA DKO +GFP-TFEB). The TFEB-transfected fibres show realignment of the lysosomes and membrane detachment (most striking in top and bottom panels) similar to those in TFEB-transfected fibres from PD mice (see Fig 4B inset). Lysosomes can be seen in the space between the fibre and plasma membrane (arrows). Bar: 10 µm for all panels.The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated autophagy-deficient Atg7:GAA DKO fibres. The increase in maximum velocity (41%) is significant (p = 2.729 × 10−18; n = 57 lysosomes for untreated; n = 52 lysosomes for TFEB-treated), but there is only a slight trend toward smaller lysosomal size (p = 7.0 × 10−2; n = 10 fibres for untreated; n = 21 fibres for TFEB-treated). The corresponding values from GAA−/− mice are presented for comparison (for TFEB-treated condition: n = 19 lysosomes for velocity measurements, and n = 10 fibres for the lysosomal size measurements). * indicates statistically significant differences (p ≤ 1.0 × 10−5 for the top panel and p ≤ 0.01 for the lower panel; Student's t-test).


A . Glycogen assay in TFEB - injected gastrocnemii and in the contralateral muscles . In TFEB - injected musclesglycogen levels were significantly decreased compared to those in untreated muscles . * indicates statistically significant differences ( p = 1 . 0 × 10 − 4 ; n = 6 ; Student ' s t - test ) .

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A. Glycogen assay in TFEB-injected gastrocnemii and in the contralateral muscles. In TFEB-injected musclesglycogen levels were significantly decreased compared to those in untreated muscles. * indicates statistically significant differences (p = 1.0 × 10−4; n = 6; Student's t-test).


B . PAS staining of TFEB - treated muscle shows a reduction of lysosomalglycogen stores ( puncta ) compared to those in untreated muscle . Original magnification : 20 × .

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B. PAS staining of TFEB-treated muscle shows a reduction of lysosomalglycogen stores (puncta) compared to those in untreated muscle. Original magnification: 20×.


C . LAMP1 staining of TFEB - injected gastrocnemii and of the contralateral untreated muscles . In TFEB - treated muscles , the size of LAMP1 - positive vesicles was reduced . Bar : 2 µm .

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C. LAMP1 staining of TFEB-injected gastrocnemii and of the contralateral untreated muscles. In TFEB-treated muscles, the size of LAMP1-positive vesicles was reduced. Bar: 2 µm.


A . EM images of muscle injected with either AAV - GFP ( untreated ) or AAV - TFEB ( TFEB - treated ) . Asterisks indicate glycogen - containing lysosomes . Bar : 1 . 5 µm ( upper panels ) and 0 . 45 µm ( lower panels ) . Higher magnification images ( lower panels ) show that glycogen particles are less densely packed in TFEB - treated muscle . Black arrows indicate autophagosome profiles ; the white empty arrow shows remnants of mitochondria engulfed by the lysosome . B . Graphical presentations of lysosomal length ( average ± SE ; n = 100 lysosomes ; p = 6 . 31 × 10 − 5 ) , the number of lysosomes per 5 µm2 area of muscle fibre section ( average ± SE ; n = 50 fields ; p = 4 . 80 × 10 − 3 ) , and the number of autophagosomes flanking glycogen - containing lysosomes ( average ± SE ; n = 100 lysosomes ; p = 4 . 39 × 10 − 5 < 0 . 001 ) . Student ' s t - test was used for each comparison . C . Graphical presentations of mitochondrial size ( average ± SE ; n = 100 ) and the number of mitochondria per 5 µm2 area of muscle fibre section ( average ± SE ; n = 50 fields ) . The differences were not significant by Student ' s t - test ( p = 3 . 65 × 10 − 1 and 4 . 27 × 10 − 1 , respectively ) .

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A. EM images of muscle injected with either AAV-GFP (untreated) or AAV-TFEB (TFEB-treated). Asterisks indicate glycogen-containing lysosomes. Bar: 1.5 µm (upper panels) and 0.45 µm (lower panels). Higher magnification images (lower panels) show that glycogen particles are less densely packed in TFEB-treated muscle. Black arrows indicate autophagosome profiles; the white empty arrow shows remnants of mitochondria engulfed by the lysosome.B. Graphical presentations of lysosomal length (average ± SE; n = 100 lysosomes; p = 6.31 × 10−5), the number of lysosomes per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields; p = 4.80 × 10−3), and the number of autophagosomes flanking glycogen-containing lysosomes (average ± SE; n = 100 lysosomes; p = 4.39 × 10−5 < 0.001). Student's t-test was used for each comparison.C. Graphical presentations of mitochondrial size (average ± SE; n = 100) and the number of mitochondria per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields). The differences were not significant by Student's t-test (p = 3.65 × 10−1 and 4.27 × 10−1, respectively).


A Phenotypes of WT ( Col - 0 ) , two gso1 mutant alleles ( gso1 - 1 , gso1 - 3 ) and two independent complementation lines ( com - 1 , com - 2 ) . Seedlings were grown on 1 / 2 MS for 6 days and then transferred to 1 / 2 MS medium supplemented with or without 100 mM NaCl for 10 days . B , C Primary root length and seedling fresh weight of seedlings depicted in ( A ) were measured at day 10 after transfer . ( mean ± SEM , n = 10 , P < 0 . 05 , two - way ANOVA )

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A Phenotypes of WT (Col-0), two gso1 mutant alleles (gso1-1, gso1-3) and two independent complementation lines (com-1, com-2). Seedlings were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days. B,C Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer. (mean ± SEM, n = 10, P < 0.05, two-way ANOVA)


A Co - IP of GSO1 and SOS2 using Arabidopsis protoplasts transiently expressing Myc - GSO1KD ( GSO1 kinase domain ) and Flag - SOS2 , Flag - HA - SOS3 or Flag - SCaBP8 . Protein extracts were immunoprecipitated with anti - Myc antibody and immunoblotted with anti - Myc antibody or anti - Flag antibody .

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A Co-IP of GSO1 and SOS2 using Arabidopsis protoplasts transiently expressing Myc-GSO1KD (GSO1 kinase domain) and Flag-SOS2, Flag-HA-SOS3 or Flag-SCaBP8. Protein extracts were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Myc antibody or anti-Flag antibody.


C High resolution localization analyses of SOS2 and GSO1 expressed under their respective native promoter at 1 . 5 mm from the quiescent center . Propidium iodide ( PI ) was used to stain the cell wall . Scale bars : 100 µm . ( BF : brightfield )

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C High resolution localization analyses of SOS2 and GSO1 expressed under their respective native promoter at 1.5 mm from the quiescent center. Propidium iodide (PI) was used to stain the cell wall. Scale bars: 100 µm. (BF: brightfield)


D Phenotypes of WT ( Col - 0 ) , gso1 - 3 , sos2 - 2 and gso1 - 3 sos2 - 2 . Seedlings were grown on 1 / 2 MS medium for 6 days and then transferred to 1 / 2 MS supplemented with or without extra NaCl for 10 days . E , F Primary root length and fresh weight of seedlings depicted in ( D ) were measured at day 7 after transfer . ( mean ± SEM , n = 10 , P < 0 . 05 , two - way ANOVA )

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D Phenotypes of WT (Col-0), gso1-3, sos2-2 and gso1-3 sos2-2. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS supplemented with or without extra NaCl for 10 days. E,F Primary root length and fresh weight of seedlings depicted in (D) were measured at day 7 after transfer. (mean ± SEM, n = 10, P < 0.05, two-way ANOVA)


A Yeast strain JP837 cells transformed with empty vectors ( control ) or the indicated combinations of Arabidopsis genes were grown on AP medium supplemented with or without 100 mM NaCl for 3 days at 28 ℃ . ( M : myristoylation motif , KD : kinase domain )

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A Yeast strain JP837 cells transformed with empty vectors (control) or the indicated combinations of Arabidopsis genes were grown on AP medium supplemented with or without 100 mM NaCl for 3 days at 28℃. (M: myristoylation motif, KD: kinase domain)


G - L Phenotypes , primary root length and seedling fresh weight of indicated plant lines grown on 1 / 2 MS medium for 6 days and then transferred to 1 / 2 MS medium supplemented with 100 mM NaCl for 7 days . ( mean ± SEM , n = 10 , P < 0 . 05 , one - way ANOVA )

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G-L Phenotypes, primary root length and seedling fresh weight of indicated plant lines grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with 100 mM NaCl for 7 days. (mean ± SEM, n = 10, P < 0.05, one-way ANOVA)


A Coomassie Brilliant Blue stain ( CBB ) and autoradiograph of in vitro kinase assay combining the indicated combinations of SOS2 / SOS2K40N and GSO1KD / GSO1KDK979N .

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A Coomassie Brilliant Blue stain (CBB) and autoradiograph of in vitro kinase assay combining the indicated combinations of SOS2/SOS2K40N and GSO1KD/GSO1KDK979N.


B Immunoblot using anti - Myc antibody and CBB and autoradiograph from kinase assays combining Myc - SOS2 with SOS1C300 as substrate . 10 - day - old seedlings stably expressing Pro35S : 6Myc - SOS2 in Col - 0 or gso1 - 3 were treated with or without 100 mM NaCl for 12 h , and Myc - SOS2 protein immunoprecipitated with anti - C - Myc antibody - conjugated agarose from roots was used in the assays . Signal strength of the phosphorylation bands has been calculated relative to the left lane .

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B Immunoblot using anti-Myc antibody and CBB and autoradiograph from kinase assays combining Myc-SOS2 with SOS1C300 as substrate. 10-day-old seedlings stably expressing Pro35S:6Myc-SOS2 in Col-0 or gso1-3 were treated with or without 100 mM NaCl for 12 h, and Myc-SOS2 protein immunoprecipitated with anti-C-Myc antibody-conjugated agarose from roots was used in the assays. Signal strength of the phosphorylation bands has been calculated relative to the left lane.


C CBB and autoradiograph of in vitro kinase assays combining GSO1KD ( kinase domain ) with SOS2K40N or SOS2T16AK40N . D CBB and autoradiograph of in vitro kinase assays using SOS2 p - site variants addressing autophosphorylation activity .

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C CBB and autoradiograph of in vitro kinase assays combining GSO1KD (kinase domain) with SOS2K40N or SOS2T16AK40N. D CBB and autoradiograph of in vitro kinase assays using SOS2 p-site variants addressing autophosphorylation activity.


A Phenotypes of WT ( Col - 0 ) , gso1 - 3 , esb1 , and casp1 casp3 . Seedlings were grown on 1 / 2 MS for 6 days and then transferred to 1 / 2 MS medium with or without 100 mM NaCl for 10 days . B , C Primary root length and seedling fresh weight of seedlings depicted in ( A ) were measured at day 10 after transfer . ( mean ± SEM , n = 12 , two - way ANOVA , P < 0 . 05 )

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A Phenotypes of WT (Col-0), gso1-3, esb1, and casp1 casp3. Seedlings were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium with or without 100 mM NaCl for 10 days. B, C Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer. (mean ± SEM, n = 12, two-way ANOVA, P < 0.05)


G Phenotypes of WT ( Col - 0 ) , cif1 cif2 , and gso1 - 3 . Seedlings were grown on 1 / 2 MS medium for 6 days and then transferred to 1 / 2 MS medium supplemented with or without 100 mM NaCl for 10 days .

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G Phenotypes of WT (Col-0), cif1 cif2, and gso1-3. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days.


J Quantification of Fluorol Yellow fluorescence intensity indicating amounts of suberin accumulation in maximum projections of z - stacks in the first mm of the continuously suberized endodermis in the indicated genotypes . Seedlings were grown on 1 / 2 MS for 5 days and then treated with the indicated NaCl concentrations for 2 days . ( mean ± SEM , n = 3 - 5 , P < 0 . 05 , one - way ANOVA and post hoc Tukey Kramer test , different letters indicate significant difference )

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J Quantification of Fluorol Yellow fluorescence intensity indicating amounts of suberin accumulation in maximum projections of z-stacks in the first mm of the continuously suberized endodermis in the indicated genotypes. Seedlings were grown on 1/2 MS for 5 days and then treated with the indicated NaCl concentrations for 2 days. (mean ± SEM, n = 3-5, P < 0.05, one-way ANOVA and post hoc Tukey Kramer test, different letters indicate significant difference)


A - D Protein accumulation ( A , C , D ) or gene expression ( B ) of SOS2 , CIF2 , GSO1 , and SGN1 displayed as false color fluorescence intensity . Seedlings from the indicated plant lines were grown on 1 / 2 MS for 4 - 5 days and then transferred to 1 / 2 MS with or without 100 mM NaCl for 1 day . Scale bars : 200 µm .

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A-D Protein accumulation (A, C, D) or gene expression (B) of SOS2, CIF2, GSO1, and SGN1 displayed as false color fluorescence intensity. Seedlings from the indicated plant lines were grown on 1/2 MS for 4-5 days and then transferred to 1/2 MS with or without 100 mM NaCl for 1 day. Scale bars: 200 µm.


E - H Longitudinal quantification of protein accumulation or gene expression depicted in ( A - D ) represented as 8 - bit fluorescence intensity . ( Mean ± SEM , n = 4 - 5 )

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E-H Longitudinal quantification of protein accumulation or gene expression depicted in (A-D) represented as 8-bit fluorescence intensity. (Mean ± SEM, n = 4-5)


( A ) In vivo fundusimage of an AAV - injected rd1mouse expressing ReaChR - mCitrine driven by the hSyn promoter ( AAV2 - hSyn : ReaChR - mCitrine ) ; transduced RGCs are visible as green puncta .

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(A) In vivo fundusimage of an AAV-injected rd1mouse expressing ReaChR-mCitrine driven by the hSyn promoter (AAV2-hSyn:ReaChR-mCitrine); transduced RGCs are visible as green puncta.


( B ) Live 2 - photon image showing that ReaChR - mCitrine expression is restricted to RGC - membranes and their dendritic processes .

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(B) Live 2-photon image showing that ReaChR-mCitrine expression is restricted to RGC-membranes and their dendritic processes.


( C ) Confocal image of a vertical retinal section expressing ReaChR - mCitrine . Endogenous mCitrine ( mCit ) fluorescence , is localized to RGCmembranes as well as RGCdendritic processes ( see also Fig EV1A ) ; blue : DAPI ( to visualize retinalnuclei ) ; inner nuclear layer ( INL ) ; inner plexiform layer ( IPL ) and ganglion cell layer ( GCL ) ; scale bars = 10 µm .

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(C) Confocal image of a vertical retinal section expressing ReaChR-mCitrine. Endogenous mCitrine (mCit) fluorescence, is localized to RGCmembranes as well as RGCdendritic processes (see also Fig EV1A); blue: DAPI (to visualize retinalnuclei); inner nuclear layer (INL); inner plexiform layer (IPL) and ganglion cell layer (GCL); scale bars = 10 µm.


( A ) Light sensitivity : Photocurrents of a ReaChR - expressing RGC stimulated with varying light intensities at 590 nm ( ~ 1013 − 1018 photons cm - 2 s - 1 ) .

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(A) Light sensitivity: Photocurrents of a ReaChR-expressing RGC stimulated with varying light intensities at 590 nm (~1013 − 1018 photons cm-2 s-1).


( B ) ReaChR - induced photocurrents as a function of light intensity ( 590 nm , n = 7 cells , mean photocurrent amplitude at 4 . 2 × 1017 photons cm - 2 s - 1 was − 349 ± 192 pA ) . At the bottom , the sensitivity range for wild - type cones ( Nikonov et al , 2006 ) is shown in comparison to the sensitivity range of ReaChR expressing RGCs . The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm , respectively ( EU - Directive , 2006 ; ICNIRP , 2013 ) .

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(B) ReaChR-induced photocurrents as a function of light intensity (590 nm, n = 7 cells, mean photocurrent amplitude at 4.2 × 1017 photons cm-2 s-1 was −349 ± 192 pA). At the bottom, the sensitivity range for wild-type cones (Nikonov et al, 2006) is shown in comparison to the sensitivity range of ReaChR expressing RGCs. The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm, respectively (EU-Directive, 2006; ICNIRP, 2013).


( C ) Photocurrent action spectrum of a ReaChR - expressing RGC ( 1 . 2 × 1016 photons cm - 2 s - 1 ) . ( D ) Normalized action spectrums of ReaChR - RGCs . Solid line represents patch - clamp data ( n = 8 cells ; 1 . 2 × 1016 photons cm - 2 s - 1 ) . The mean photocurrent amplitude at 550 nm was − 324 ± 97 pA . Dashed line represents multi - electrode array data ( n = 168 cells ; ~ 1016 photons cm - 2 s - 1 ) .

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(C) Photocurrent action spectrum of a ReaChR-expressing RGC (1.2 × 1016 photons cm-2 s-1). (D) Normalized action spectrums of ReaChR-RGCs. Solid line represents patch-clamp data (n = 8 cells; 1.2 × 1016 photons cm-2 s-1). The mean photocurrent amplitude at 550 nm was −324 ± 97 pA. Dashed line represents multi-electrode array data (n = 168 cells; ~1016 photons cm-2 s-1).


( E ) Temporal properties . Modulation of ReaChR - induced photocurrents at increasing stimulation frequencies ( patch - clamp recording from a ReaChR - expressing RGC ; at 22 Hz magnified trace is shown ; 1 . 3 × 1016 photons cm - 2 s - 1 ) . ( F ) Spiking modulation ratio of ReaChR - RGCs quantified from multi - electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz ( 297 cells , 1 . 3 × 1016 photons cm - 2 s - 1 ) .

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(E) Temporal properties. Modulation of ReaChR-induced photocurrents at increasing stimulation frequencies (patch-clamp recording from a ReaChR-expressing RGC; at 22 Hz magnified trace is shown; 1.3×1016 photons cm-2 s-1). (F) Spiking modulation ratio of ReaChR-RGCs quantified from multi-electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz (297 cells, 1.3 × 1016 photons cm-2 s-1).


( A ) Examples of local field potential ( LFP ) traces in response to light flashes at 595 nm ( light stimulus is shown as orange bar ; 1 . 25 × 1017 photons cm - 2 s - 1 ) . ( B ) Spike raster plots in response to 200 repetitions of the same light stimulus . ( C ) Averaged spike responses shown as peristimulus time histograms ( PSTH , bin size : 5 ms ) based on the spike raster plots . Top - row : WT - mice , middle row : rd1 control mice , bottom - row : ReaChR - treated rd1 mice . ( D ) ReaChR - induced cortical responses ( spikes and LFP ) as a function of light intensity at ( 595 nm ) demonstrating that ReaChR - treated rd1 mice ( n = 4 ) can detect light in the visual cortex at intensities well below the safety threshold allowed for the humanretina . Data presented as mean ± SEM .

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(A) Examples of local field potential (LFP) traces in response to light flashes at 595 nm (light stimulus is shown as orange bar; 1.25 × 1017 photons cm-2 s-1). (B) Spike raster plots in response to 200 repetitions of the same light stimulus. (C) Averaged spike responses shown as peristimulus time histograms (PSTH, bin size: 5 ms) based on the spike raster plots. Top-row: WT-mice, middle row: rd1 control mice, bottom-row: ReaChR-treated rd1 mice. (D) ReaChR-induced cortical responses (spikes and LFP) as a function of light intensity at (595 nm) demonstrating that ReaChR-treated rd1 mice (n = 4) can detect light in the visual cortex at intensities well below the safety threshold allowed for the humanretina. Data presented as mean ± SEM.


( A ) Locomotory behavior of a representative rd1 and ReaChR - treated mouse during illumination with light at 590 nm in an open field arena ( irradiance measured at the level of the mice ' s eyes was ~ 1015 photons cm - 2 s - 1 , cumulative activity trajectories are shown as red traces after 2 min of illumination ) , see also EV Movie 1 and EV Movie 2 . ( B ) Mean velocities of the ReaChR - treated and rd1mice analyzed over a duration of 2 min at light onset . The mean velocity of ReaChR - treated mice were significantly reduced than rd1mice ( P = 0 . 006 , one - tailed t - test ) upon illumination . Mean ± SEM are shown for each group . ReaChR - treated rd1 ( n = 11 ) , rd1 ( n = 12 ) .

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(A) Locomotory behavior of a representative rd1 and ReaChR-treated mouse during illumination with light at 590 nm in an open field arena (irradiance measured at the level of the mice's eyes was ~1015 photons cm-2 s-1, cumulative activity trajectories are shown as red traces after 2 min of illumination), see also EV Movie 1 and EV Movie 2. (B) Mean velocities of the ReaChR-treated and rd1mice analyzed over a duration of 2 min at light onset. The mean velocity of ReaChR-treated mice were significantly reduced than rd1mice (P = 0.006, one-tailed t-test) upon illumination. Mean ± SEM are shown for each group. ReaChR-treated rd1 (n = 11), rd1 (n = 12).


( C ) Light / dark box test . Percentage of time mice spent in the light compartment as a function of light intensity ( WT , n = 9 ; rd1 , n = 9 ; ReaChR - treated n = 7 ) . Mean ± SEM are shown for each group . ( D ) Percentage of time rd1 ( n = 9 ) , WT ( n = 9 ) and ReaChR - treated ( n = 7 ) mice spent in the light compartment for an illumination intensity of 2 . 11 × 1015 photons cm - 2 s - 1 . Both WT as well as ReaChR - treated mice spent significantly less time in the light compartment than the rd1mice ( P < 0 . 0001 , ordinary one - way ANOVA ) . * * * and * * * * indicates significance levels determined by Tukey ' s multiple comparisons test for rd1 vs . WT and rd1 vs . ReaChR - treated . See also EV Movie 3 for the response of a representative ReaChR - treated rd1 mouse plotted in ( D ) .

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(C) Light/dark box test. Percentage of time mice spent in the light compartment as a function of light intensity (WT, n = 9; rd1, n = 9; ReaChR-treated n = 7). Mean ± SEM are shown for each group. (D) Percentage of time rd1 (n = 9), WT (n = 9) and ReaChR-treated (n = 7) mice spent in the light compartment for an illumination intensity of 2.11 × 1015 photons cm-2 s-1. Both WT as well as ReaChR-treated mice spent significantly less time in the light compartment than the rd1mice (P < 0.0001, ordinary one-way ANOVA). *** and **** indicates significance levels determined by Tukey's multiple comparisons test for rd1 vs. WT and rd1 vs. ReaChR-treated. See also EV Movie 3 for the response of a representative ReaChR-treated rd1 mouse plotted in (D).


( A ) Vertical section of macaqueretinal explant transfected with AAV2 hSyn : ReaChR - mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL . ( B ) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F - CAG : ReaChR - GFP after 3 days of incubation . GFP expression was primarily observed in the cell bodies but also in processes of RGCs ; green : endogenous GFP fluorescence ; blue : DAPI ; outer nuclear layer ( ONL ) . scale bars = 10 µm .

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(A) Vertical section of macaqueretinal explant transfected with AAV2 hSyn:ReaChR-mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL. (B) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F-CAG:ReaChR-GFP after 3 days of incubation. GFP expression was primarily observed in the cell bodies but also in processes of RGCs; green: endogenous GFP fluorescence; blue: DAPI; outer nuclear layer (ONL). scale bars = 10 µm.


( C ) Averaged spike responses obtained from multi - electrode array recordings shown as PSTH of 4 , 8 and 2 cells respectively of three different macaques ( 580 nm ; 1 . 24 × 1017 photons cm - 2 s - 1 ) . PSTHs are presented as mean ± SEM ( dashed lines , calculated over responding cells ) . Raster plots of a representative cell from each individual macaque are shown below ( 10 stimulus - repetitions ) .

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(C) Averaged spike responses obtained from multi-electrode array recordings shown as PSTH of 4, 8 and 2 cells respectively of three different macaques (580 nm; 1.24 × 1017 photons cm-2 s-1). PSTHs are presented as mean ± SEM (dashed lines, calculated over responding cells). Raster plots of a representative cell from each individual macaque are shown below (10 stimulus-repetitions).


( D ) Action spectrum of ReaChR in primate RGCs from multi - electrode array recordings . Data is shown as mean ± SEM of the normalized firing rate , averaged from three individual macaques ( 28 cells , error bars calculated over cells ) , see also Fig EV2B for action spectrums of three individual macaques obtained from multi - electrode array recordings . ( E ) Responses ( Hz ) of a ReaChR - expressing RGC ( patch clamp recording , in cell - attached mode ) to flicker stimulations at increasing frequencies ( 2 − 22 Hz ; 1 . 3 × 1016 photons cm - 2 s - 1 ) .

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(D) Action spectrum of ReaChR in primate RGCs from multi-electrode array recordings. Data is shown as mean ± SEM of the normalized firing rate, averaged from three individual macaques (28 cells, error bars calculated over cells), see also Fig EV2B for action spectrums of three individual macaques obtained from multi-electrode array recordings. (E) Responses (Hz) of a ReaChR-expressing RGC (patch clamp recording, in cell-attached mode) to flicker stimulations at increasing frequencies (2−22 Hz; 1.3 × 1016 photons cm-2 s-1).


( B ) Brightfield image of the retinal explant showing the macula ( yellow pigmented ) with the approximate location of the multi - electrode array chip indicated .

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(B) Brightfield image of the retinal explant showing the macula (yellow pigmented) with the approximate location of the multi-electrode array chip indicated.


( C ) Infrared image of the explant pressed against the multi - electrode array ; the black dashed line depicts the ' electrophysiological boundary ' of the para - fovea based on the absence of RGC - spike trains ( scale bar : 100 µm ; see also Fig EV3A and B ) . Voltage trace of a para - foveal RGC stimulated with orange light ( 600 nm ; 1 . 1 × 1017 photons cm - 2 s - 1 ) is shown on the right . Individual electrodes that recorded ReaChR - responding cells are indicated in green .

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(C) Infrared image of the explant pressed against the multi-electrode array; the black dashed line depicts the 'electrophysiological boundary' of the para-fovea based on the absence of RGC-spike trains (scale bar: 100 µm; see also Fig EV3A and B). Voltage trace of a para-foveal RGC stimulated with orange light (600 nm; 1.1 × 1017 photons cm-2 s-1) is shown on the right. Individual electrodes that recorded ReaChR-responding cells are indicated in green.


( D ) Action spectrum of ReaChR constructed from ( 6 cells , mean ± SEM , error bars calculated over cells ) exhibiting peak activity at 550 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) similar to what was observed in mice and in the macaqueretinae .

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(D) Action spectrum of ReaChR constructed from (6 cells, mean ± SEM, error bars calculated over cells) exhibiting peak activity at 550 nm (1.1 × 1017 photons cm-2 s-1) similar to what was observed in mice and in the macaqueretinae.


( E ) Peristimulus time histograms constructed for wavelengths 400 to 650 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) demonstrate ReaChR - evoked spike trains ( under L - AP4 ) in the humanretina ( 6 cells , mean ± SEM , error bars calculated over cells ) , bin size : 100 ms ; Bottom : Raster plots of the light responses of a representative spike - sorted cell .

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(E) Peristimulus time histograms constructed for wavelengths 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate ReaChR-evoked spike trains (under L-AP4) in the humanretina (6 cells, mean ± SEM, error bars calculated over cells), bin size: 100 ms; Bottom: Raster plots of the light responses of a representative spike-sorted cell.


( A ) Confocal image of the central retina , exhibiting endogenous ReaChR - mCitrine fluorescence ( mCit ) in ganglion cells , located within ~ 500 µm from the center of the foveal pit , a part of the humanretina where midget cell density is estimated to be ~ 95 % of total RGC population ( Dacey , 1993 ) ( arrowhead : soma ; arrow : dendritic arbor ) . The dashed line illustrates the beginning of the foveal slope leading towards the ' cone - only ' foveal pit . ( B ) Confocal image of the same explant at a higher retinal eccentricity ( 3 - 4 mm from the foveal center ) showing ReaChR - mCitrine fluorescence in RGCs with larger dendritic arbors . The endogenous mCitrine was not amplified . Scale bars : 10 µm . PR : Photoreceptors , GC : Ganglion cells , DAPI ( blue ) .

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(A) Confocal image of the central retina, exhibiting endogenous ReaChR-mCitrine fluorescence (mCit) in ganglion cells, located within ~500 µm from the center of the foveal pit, a part of the humanretina where midget cell density is estimated to be ~95% of total RGC population (Dacey, 1993) (arrowhead: soma; arrow: dendritic arbor). The dashed line illustrates the beginning of the foveal slope leading towards the 'cone-only' foveal pit. (B) Confocal image of the same explant at a higher retinal eccentricity (3-4 mm from the foveal center) showing ReaChR-mCitrine fluorescence in RGCs with larger dendritic arbors. The endogenous mCitrine was not amplified. Scale bars: 10 µm. PR: Photoreceptors, GC: Ganglion cells, DAPI (blue).


( A ) Humanretinal explant , prepared from the far periphery , infected with an AAV8 Y447 733F - CAG : ReaChR - GFP , placed on a multi - electrode array chip ( left ) ; GFP epifluorescence of the same explant following 12 days of incubation is shown on the right . Scale bars : 100 µm . The location of two cells that responded to repeated 2 s full field stimulations of orange light ( 600 nm ) are highlighted .

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(A) Humanretinal explant, prepared from the far periphery, infected with an AAV8 Y447 733F-CAG :ReaChR-GFP, placed on a multi-electrode array chip (left); GFP epifluorescence of the same explant following 12 days of incubation is shown on the right. Scale bars: 100 µm. The location of two cells that responded to repeated 2 s full field stimulations of orange light (600 nm) are highlighted.


( B ) The corresponding voltage traces show that the responses in the far peripheral humanretina are more transient , compared to the experiments from the human para - fovea ( see Fig 6C and E , Fig EV3B ) where only sustained light responses were evoked . The light response of cell ( 2 ) was restricted to a burst of action potentials fired within the initial ~ 300 ms in response to a 2 s full field 600 nm light stimulus .

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(B) The corresponding voltage traces show that the responses in the far peripheral humanretina are more transient, compared to the experiments from the human para-fovea (see Fig 6C and E, Fig EV3B) where only sustained light responses were evoked. The light response of cell (2) was restricted to a burst of action potentials fired within the initial ~300 ms in response to a 2 s full field 600 nm light stimulus.


( C ) Peristimulus time histograms constructed for wavelengths from 400 to 650 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) demonstrate AAV - ReaChR - evoked spike trains ( mean ± SEM , error bars calculated over 3 - 4 cells , bin size : 100 ms ) Bottom : Raster plots of the light responses of a representative spike - sorted cell . All recordings were performed under pharmacological blockade of photoreceptor input ( 50 µM L - AP4 ) .

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(C) Peristimulus time histograms constructed for wavelengths from 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate AAV-ReaChR-evoked spike trains (mean ± SEM, error bars calculated over 3-4 cells, bin size: 100 ms) Bottom: Raster plots of the light responses of a representative spike-sorted cell. All recordings were performed under pharmacological blockade of photoreceptor input (50 µM L-AP4).


A . Histograms of crossovers ( blue ) per F2 individual in the indicated populations , with the Poisson expectation plotted in red . Mean values are indicated by the black dotted lines . The genome average SNPs / kb for each cross are printed above the plots .

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A. Histograms of crossovers (blue) per F2 individual in the indicated populations, with the Poisson expectation plotted in red. Mean values are indicated by the black dotted lines. The genome average SNPs/kb for each cross are printed above the plots.


B . Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes . Mean values are shown by horizontal dashed lines . SNPs per 200 kb are plotted and shaded in colour ( Col × Ws ( purple ) , Col × Bur ( Lawrence et al , 2019 ) ( dark green ) , Col × Ct ( light blue ) , Col × Ler F2 populations with 17 , 077 ( red ) and 1 , 840 ( blue ) crossovers ( Serra et al , 2018b ; Rowan et al , 2019 ) and Col × CLC ( pink / light green , where pink indicates Cvi SNPs and green indicates Ler SNPs ) ) . The positions of telomeres ( TEL ) and centromeres ( CEN ) are indicated . The number of crossovers analysed is printed inset . C . Data as for B , but analyzing crossovers ( lines ) or SNPs ( shading ) along proportionally scaled chromosome arms , orientated from telomere ( TEL ) to centromere ( CEN ) .

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B. Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes. Mean values are shown by horizontal dashed lines. SNPs per 200 kb are plotted and shaded in colour (Col×Ws (purple), Col×Bur (Lawrence et al, 2019) (dark green), Col×Ct (light blue), Col×Ler F2 populations with 17,077 (red) and 1,840 (blue) crossovers (Serra et al, 2018b; Rowan et al, 2019) and Col×CLC (pink/light green, where pink indicates Cvi SNPs and green indicates Ler SNPs)). The positions of telomeres (TEL) and centromeres (CEN) are indicated. The number of crossovers analysed is printed inset. C. Data as for B, but analyzing crossovers (lines) or SNPs (shading) along proportionally scaled chromosome arms, orientated from telomere (TEL) to centromere (CEN).


A . Crossover frequency normalised by the number of F2 individuals , and SNP density in 100 kilobase ( kb ) adjacent windows were calculated for each population and ranked into percentiles according to SNP density . Centromeric regions were excluded from analysis ( Underwood et al , 2018 ) . The Spearman ' s rank correlation coefficient ( r ) between SNP density and crossover frequency is printed inset . Trend lines were fitted in ggplot using a generalized additive model ( GAM ) with the formula y ~ poly ( x , 2 ) .

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A. Crossover frequency normalised by the number of F2 individuals, and SNP density in 100 kilobase (kb) adjacent windows were calculated for each population and ranked into percentiles according to SNP density. Centromeric regions were excluded from analysis (Underwood et al, 2018). The Spearman's rank correlation coefficient (r) between SNP density and crossover frequency is printed inset. Trend lines were fitted in ggplot using a generalized additive model (GAM) with the formula y~poly(x,2).


A . SNPs / kb in physical windows of increasing size ( kb ) around crossover midpoints ( red ) , or for matched randomly chosen positions ( grey ) . Printed above the plot for each window are circles coloured green if crossover SNPs / kb values are significantly different to random ( P < 0 . 05 ) , or red if not ( P > 0 . 05 ) ( Bonferroni adjusted t - tests ) . The population ( Col × Ws , Col × Bur ( Lawrence et al , 2019 ) , Col × Ct , Col × Ler ( Serra et al , 2018b ; Rowan et al , 2019 ) and Col × CLC ) is printed above each plot , and the number of positions analysed is printed inset .

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A. SNPs/kb in physical windows of increasing size (kb) around crossover midpoints (red), or for matched randomly chosen positions (grey). Printed above the plot for each window are circles coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population (Col×Ws, Col×Bur (Lawrence et al, 2019), Col×Ct, Col×Ler (Serra et al, 2018b; Rowan et al, 2019) and Col×CLC) is printed above each plot, and the number of positions analysed is printed inset.


B . As for A , but analyzing the indicated Col / Ler transition and transversion polymorphisms around Col × Ler crossovers ( Rowan et al , 2019 ) ( red ) , or the same number of random positions ( grey ) .

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B. As for A, but analyzing the indicated Col/Ler transition and transversion polymorphisms around Col×Ler crossovers (Rowan et al, 2019) (red), or the same number of random positions (grey).


C . As for A , but analysing Col / Ler SNPs / kb around crossovers from wild type , HEI10 - OE , recq4a recq4b or HEI10 - OE recq4a recq4b ( Serra et al , 2018b ) . All populations were generated from Col × Ler hybrids . Printed beneath are the estimated average numbers of Class I and Class II crossovers per meiosis for each genotype , measured via genotyping - by - sequencing ( Serra et al , 2018b ; Ziolkowski et al , 2017 ) . To the right are plots showing the significance of SNPs / kb differences between crossover sets , across the windows tested ( Bonferroni adjusted t - tests ) .

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C. As for A, but analysing Col/Ler SNPs/kb around crossovers from wild type, HEI10-OE, recq4a recq4b or HEI10-OE recq4a recq4b (Serra et al, 2018b). All populations were generated from Col×Ler hybrids. Printed beneath are the estimated average numbers of Class I and Class II crossovers per meiosis for each genotype, measured via genotyping-by-sequencing (Serra et al, 2018b; Ziolkowski et al, 2017). To the right are plots showing the significance of SNPs/kb differences between crossover sets, across the windows tested (Bonferroni adjusted t-tests).


A . Histograms of observed crossovers per F2 individual in the indicated wild type and msh2 populations , from Col × Ct , Col × Ler and Col × CLC crosses ( blue ) . The Poisson expectation is plotted in red . Data from HEI10 - OE , recq4a recq4b and HEI10 - OE recq4a recq4b F2 populations are also shown for comparison ( Serra et al , 2018b ; Ziolkowski et al , 2017 ) . Mean values are indicated by black dotted lines .

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A. Histograms of observed crossovers per F2 individual in the indicated wild type and msh2 populations, from Col×Ct, Col×Ler and Col×CLC crosses (blue). The Poisson expectation is plotted in red. Data from HEI10-OE, recq4a recq4b and HEI10-OE recq4a recq4b F2 populations are also shown for comparison (Serra et al, 2018b; Ziolkowski et al, 2017). Mean values are indicated by black dotted lines.


B . Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes , with mean values shown by horizontal dashed lines . Data are shown for wild type ( red ) and msh2 ( blue ) crossovers generated from Col × Ct , Col × Ler and Col × CLC hybrids . SNPs per 200 kb are shaded in colour ( blue = Col / Ct , green = Col / Ler , pink = Col / Cvi ) . The positions of telomeres ( TEL ) and centromeres ( CEN ) are labeled . The number of crossovers analysed is printed inset . C . Data as for B , but analyzing crossovers in wild type ( red ) and msh2 ( blue ) , or SNPs along proportionally scaled chromosome arms orientated from telomeres ( TEL ) to centromeres ( CEN ) . Crossover data from HEI10 - OE , recq4a recq4b and HEI10 - OE recq4a recq4b Col × Ler F2 populations are also shown ( Serra et al , 2018b ; Ziolkowski et al , 2017 ) .

O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

B. Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes, with mean values shown by horizontal dashed lines. Data are shown for wild type (red) and msh2 (blue) crossovers generated from Col×Ct, Col×Ler and Col×CLC hybrids. SNPs per 200 kb are shaded in colour (blue=Col/Ct, green=Col/Ler, pink=Col/Cvi). The positions of telomeres (TEL) and centromeres (CEN) are labeled. The number of crossovers analysed is printed inset. C. Data as for B, but analyzing crossovers in wild type (red) and msh2 (blue), or SNPs along proportionally scaled chromosome arms orientated from telomeres (TEL) to centromeres (CEN). Crossover data from HEI10-OE, recq4a recq4b and HEI10-OE recq4a recq4b Col×Ler F2 populations are also shown (Serra et al, 2018b; Ziolkowski et al, 2017).


A . Histograms showing the mean distance ( megabases , Mb ) of observed double crossovers ( DCO , red vertical line ) , compared to 2 , 000 matched sets of randomly generated distances ( RAN , black ) . The mean distance of the random sets is shown in by the vertical grey line .

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A. Histograms showing the mean distance (megabases, Mb) of observed double crossovers (DCO, red vertical line), compared to 2,000 matched sets of randomly generated distances (RAN, black). The mean distance of the random sets is shown in by the vertical grey line.


B . SNPs / kb values were calculated in physical windows of increasing size ( kb ) around crossover midpoints in wild type ( red ) and msh2 ( blue ) . The same values were calculated for matched randomly chosen positions ( grey ) . Printed above the plot for each window are circles that are coloured green if crossover SNPs / kb values are significantly different to random ( P < 0 . 05 ) , or red if not ( P > 0 . 05 ) ( Bonferroni adjusted t - tests ) . The population analysed ( Col × Ct , Col × Ler , Col × CLC ) and its genome average SNP frequency ( SNPs / kb ) is printed above the plots and the number of crossover positions analysed is printed inset . Beneath are plots showing the significance of SNPs / kb differences around crossovers between the indicated genotypes .

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B. SNPs/kb values were calculated in physical windows of increasing size (kb) around crossover midpoints in wild type (red) and msh2 (blue). The same values were calculated for matched randomly chosen positions (grey). Printed above the plot for each window are circles that are coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population analysed (Col×Ct, Col×Ler, Col×CLC) and its genome average SNP frequency (SNPs/kb) is printed above the plots and the number of crossover positions analysed is printed inset. Beneath are plots showing the significance of SNPs/kb differences around crossovers between the indicated genotypes.


A . Representative DAPI - stained spreads of pachytene , diakinesis , metaphase - I , dyad and tetrad meiotic stages , for wild type and msh2 , in Col × Ler or Col × CLC hybrid backgrounds .

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A. Representative DAPI-stained spreads of pachytene, diakinesis, metaphase-I, dyad and tetrad meiotic stages, for wild type and msh2, in Col×Ler or Col×CLC hybrid backgrounds.


B . Male meiocytes immunostained for MSH2 ( red ) and ASY1 ( green ) , and stained for DAPI ( blue ) in wild type ( Col ) or msh2 .

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B. Male meiocytes immunostained for MSH2 (red) and ASY1 (green), and stained for DAPI (blue) in wild type (Col) or msh2.


C . As for B , but immunostaining wild type male meiocytes for MSH2 ( red ) , MSH4 ( green ) and staining chromatin with DAPI ( blue ) .

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C. As for B, but immunostaining wild type male meiocytes for MSH2 (red), MSH4 (green) and staining chromatin with DAPI (blue).


A . Crossover frequency ( black ) and SNP density ( purple ) per 200 kb measured from the Col × Ct F2 population . The position of the centromere ( CEN , vertical dashed line ) and 420 FTL transgenes ( red and green vertical lines ) used to measure crossover frequency are indicated .

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A. Crossover frequency (black) and SNP density (purple) per 200 kb measured from the Col×Ct F2 population. The position of the centromere (CEN, vertical dashed line) and 420 FTL transgenes (red and green vertical lines) used to measure crossover frequency are indicated.


B . Plots of chromosome 3 showing positions of Col / Col ( black ) or Col / Ct ( purple ) homozygous or heterozygous genotypes at the indicated marker positions ( circles ) . The positions of the 420 FTL transgenes are indicated ( red and green vertical lines ) .

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B. Plots of chromosome 3 showing positions of Col/Col (black) or Col/Ct (purple) homozygous or heterozygous genotypes at the indicated marker positions (circles). The positions of the 420 FTL transgenes are indicated (red and green vertical lines).


C . 420 crossover frequency ( cM ) in the HOM - HOM , HET - HET , HET - HOM , HOM - HET genotypes shown in B , in either wild type or msh2 . Values for replicate individuals are plotted , in addition to the mean ( red ) .

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C. 420 crossover frequency (cM) in the HOM-HOM, HET-HET, HET-HOM, HOM-HET genotypes shown in B, in either wild type or msh2. Values for replicate individuals are plotted, in addition to the mean (red).


D . As for C , but comparing genotypes in wild type or HEI10 - OE backgrounds . Values for replicate individuals are plotted , in addition to the mean ( red ) .

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D. As for C, but comparing genotypes in wild type or HEI10-OE backgrounds. Values for replicate individuals are plotted, in addition to the mean (red).


D . Plots of SNP density per 10 kb along the chromosomes for Arabidopsis Col × Ler and budding yeast S288c × SK1 crosses ( Cooper et al , 2018 ) . Telomere positions are shown by vertical lines , and centromeres by vertical dashed lines . Centromeres are also labeled above the plots in red , as ' CEN ' or ' C ' .

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D. Plots of SNP density per 10 kb along the chromosomes for Arabidopsis Col×Ler and budding yeast S288c×SK1 crosses (Cooper et al, 2018). Telomere positions are shown by vertical lines, and centromeres by vertical dashed lines. Centromeres are also labeled above the plots in red, as 'CEN' or 'C'.


( A ) The list of co - precipitated proteins as determined by immunoprecipitation followed by mass spectrometry ( IP - MS ) . The SWR1 subunits not shared by the ISWI complexes are shown in blue ; The DDT domain - containing ISWI specific subunits are shown in orange ; CHR11 and CHR17 are shared subunits of the SWR1 and ISWI complexes and are shown in green .

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(A) The list of co-precipitated proteins as determined by immunoprecipitation followed by mass spectrometry (IP-MS). The SWR1 subunits not shared by the ISWI complexes are shown in blue; The DDT domain-containing ISWI specific subunits are shown in orange; CHR11 and CHR17 are shared subunits of the SWR1 and ISWI complexes and are shown in green.


( C ) Determination of the interaction of MBD9 with HTA9 , HTA11 , PIE1 , CHR11 , and CHR17 by co - immunoprecipitation .

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(C) Determination of the interaction of MBD9 with HTA9, HTA11, PIE1, CHR11, and CHR17 by co-immunoprecipitation.


Determination of the interaction of PIE1 - Flag , MBD9 - Myc , and CHR11 - HA by in vitro pull down assays . PIE1 - Flag , MBD9 - Myc , and CHR11 - HA were expressed from yeast and subjected to the pull down assays . Anti - Flag agaroses were independently used in the pull down assays .

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Determination of the interaction of PIE1-Flag, MBD9-Myc, and CHR11-HA by in vitro pull down assays. PIE1-Flag, MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. Anti-Flag agaroses were independently used in the pull down assays.


C ) Determination of the interaction of MBD9 - Myc , and CHR11 - HA by in vitro pull down assays . MBD9 - Myc , and CHR11 - HA were expressed from yeast and subjected to the pull down assays . anti - HA ( C ) agaroses were independently used in the pull down assays .

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C) Determination of the interaction of MBD9-Myc, and CHR11-HA by in vitro pull down assays. MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. anti-HA (C) agaroses were independently used in the pull down assays.


D ) he effect of the pie1 and mbd9 - 1 mutations on the formation of the SWR1 complex as determined by IP - MS . Transgenic plants expressing Flag - tagged MBD9 , PIE1 , and CHR11 in the WT and mutant backgrounds were subjected to the IP - MS experiment with anti - Flag agarose .

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D) he effect of the pie1 and mbd9-1 mutations on the formation of the SWR1 complex as determined by IP-MS. Transgenic plants expressing Flag-tagged MBD9, PIE1, and CHR11 in the WT and mutant backgrounds were subjected to the IP-MS experiment with anti-Flag agarose.


( A ) Morphological phenotypes of the WT , chr11 / 17 , pie1 , swc2 , and rin2 mutant plants . Shown are 24 - day - old plants . The top panel shows the phenotypes of plants in the soil ; the bottom panel shows the phenotypes of plants on the black cloth .

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(A) Morphological phenotypes of the WT, chr11/17, pie1, swc2, and rin2 mutant plants. Shown are 24-day-old plants. The top panel shows the phenotypes of plants in the soil; the bottom panel shows the phenotypes of plants on the black cloth.


( B ) Shown are rosette leaves of the WT , pie1 , swc2 , and rin2 mutant plants . Rosette leaves were cut from bolting plants .

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(B) Shown are rosette leaves of the WT, pie1, swc2, and rin2 mutant plants. Rosette leaves were cut from bolting plants.