Figure 3Depicted are the fractions of SVs without filaments , ribbon - attached SVs ( Ri . ) , interconnected SVs ( In . ) and SVs with Ri . + In . per ribbon , at ( A ) the distal ( Ds . ) and ( B ) the proximal ( Pr . ) part of the ribbon . Data are represented as mean ± SEM of wild - type and OtofPga / Pga IHCs under inhib , rest and stim . ( A ) Upon stimulation , in the Ds . part at wild - type , the fraction of SVs without filaments reduces , but of SVs with Ri . + In . increases significantly . At OtofPga / Pga IHCs the fraction of SVs without filaments is reduced when comparing stim vs . inhib . The fraction of interconnected SVs increases with stimulation compared to inhibition and rest . In the mutant ( inhib ) , the fraction of interconnected SVs decreases compared to wild - type ( inhib ) , and Ri . + In . SVs reduce vs . wild - type ( stim ) . ( B ) The fraction of SVs without filaments increased at inhibited OtofPga / Pga synapses . The red dotted lines in ( A ) and ( B ) represent the mean fraction of RA - SVs / ribbon part of resting wild - type for each defined sub - pool .

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

Figure 3Depicted are the fractions of SVs without filaments, ribbon-attached SVs (Ri.), interconnected SVs (In.) and SVs with Ri. + In. per ribbon, at (A) the distal (Ds.) and (B) the proximal (Pr.) part of the ribbon. Data are represented as mean ± SEM of wild-type and OtofPga/Pga IHCs under inhib, rest and stim. (A) Upon stimulation, in the Ds. part at wild-type, the fraction of SVs without filaments reduces, but of SVs with Ri. + In. increases significantly. At OtofPga/Pga IHCs the fraction of SVs without filaments is reduced when comparing stim vs. inhib. The fraction of interconnected SVs increases with stimulation compared to inhibition and rest. In the mutant (inhib), the fraction of interconnected SVs decreases compared to wild-type (inhib), and Ri. + In. SVs reduce vs. wild-type (stim). (B) The fraction of SVs without filaments increased at inhibited OtofPga/Pga synapses. The red dotted lines in (A) and (B) represent the mean fraction of RA-SVs/ribbon part of resting wild-type for each defined sub-pool.


Figure 4Representative wild - type virtual section under rest depicts SVs with r - attached fil ( Ra . fil . , red arrowheads ) and one interconnector ( Intercon . , purple arrowhead ) , around the ribbon ( R ) . Magnification 12 , 000x ; scale bar , 40 nm . Example 3D reconstruction of the RA - SVs with filaments ( green ) in resting wild - type . Larger average length is found for ribbon - attached filaments ( Ra . fil . , red ) vs . SV interconnectors ( Intercon . , purple ) . For clear visualization , the ribbon ( R ) is transparent and the same SV pair is shown in a front ( A ' ) , side ( A ' ' ) and top view ( A ' ' ' ) . Scale bar , 50 nm . Increased mean length of ribbon - attached filaments vs . SV interconnectors in all conditions for both genotypes . The red dotted lines represent the mean RA - filament length in resting wild - type for each sub - pool . The cumulative probability distribution of RA - SVs plotted against the individual filament length . The graph illustrates shorter filaments in wild - type and OtofPga / Pga ribbon synapses upon stimulation . Decreased mean filament length upon stimulation in wild - type and OtofPga / Pga ribbon synapses . Resting OtofPga / Pga exhibit the shortest mean filament length . The red dotted lines represent the mean RA - filament length in resting wild - type for each sub - pool .

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

Figure 4Representative wild-type virtual section under rest depicts SVs with r-attached fil (Ra.fil., red arrowheads) and one interconnector (Intercon., purple arrowhead), around the ribbon (R). Magnification 12,000x; scale bar, 40 nm. Example 3D reconstruction of the RA-SVs with filaments (green) in resting wild-type. Larger average length is found for ribbon-attached filaments (Ra.fil., red) vs. SV interconnectors (Intercon., purple). For clear visualization, the ribbon (R) is transparent and the same SV pair is shown in a front (A'), side (A'') and top view (A'''). Scale bar, 50 nm.Increased mean length of ribbon-attached filaments vs. SV interconnectors in all conditions for both genotypes. The red dotted lines represent the mean RA-filament length in resting wild-type for each sub-pool.The cumulative probability distribution of RA-SVs plotted against the individual filament length. The graph illustrates shorter filaments in wild-type and OtofPga/Pga ribbon synapses upon stimulation.Decreased mean filament length upon stimulation in wild-type and OtofPga/Pga ribbon synapses. Resting OtofPga/Pga exhibit the shortest mean filament length. The red dotted lines represent the mean RA-filament length in resting wild-type for each sub-pool.


Figure 5Representative tomographic virtual sections from resting wild - type . Five different types of MP - SVs are observed that we classify as sub - pools : non - tethered SVs ( A ' , A ' ' ; yellow ) , single presynaptic density - attached SVs ( B ' , B ' ' ; Pd . a , orange , pink arrowhead ) or single m - attached SVs ( C ' , C ' ' ; Ma . , orange , orange arrowhead ) , or multiple - tethered SVs ( D ' , D ' ' ; orange , brown arrowheads ) and docked SVs ( E ' , E ' ' ; light orange ) . Ribbon ( R ) , presynaptic density ( PD , pink ) and AZ - membrane ( blue ) . For clarity , a simplified illustration is provided for each sub - pool . Magnification : 12 , 000x ; scale bar , 40 nm . The graph is showing the percentage of the MP sub - pools shown in A - E of all MP - SVs at inhibitory , resting and stimulatory wild - type synapses .

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

Figure 5Representative tomographic virtual sections from resting wild-type. Five different types of MP-SVs are observed that we classify as sub-pools: non-tethered SVs (A',A''; yellow), single presynaptic density-attached SVs (B',B''; Pd.a, orange, pink arrowhead) or single m-attached SVs (C',C''; Ma., orange, orange arrowhead), or multiple-tethered SVs (D',D''; orange, brown arrowheads) and docked SVs (E',E''; light orange). Ribbon (R), presynaptic density (PD, pink) and AZ-membrane (blue). For clarity, a simplified illustration is provided for each sub-pool. Magnification: 12,000x; scale bar, 40 nm. The graph is showing the percentage of the MP sub-pools shown in A-E of all MP-SVs at inhibitory, resting and stimulatory wild-type synapses.


Figure 6Depicts the fraction of non - tethered and single - tethered MP - SVs per ribbon . Data are presented in mean ± SEM . The fraction of non - tethered SVs reduces in wild - type and OtofPga / Pga after stimulation and the fraction of single membrane - attached tethers increases in wild - type upon stimulation . In resting wild - type , most single - tethered SVs are attached to the presynaptic density .

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 6Depicts the fraction of non-tethered and single-tethered MP-SVs per ribbon. Data are presented in mean ± SEM. The fraction of non-tethered SVs reduces in wild-type and OtofPga/Pga after stimulation and the fraction of single membrane-attached tethers increases in wild-type upon stimulation. In resting wild-type, most single-tethered SVs are attached to the presynaptic density.


Figure 7Example tomographic virtual sections from inhibitory : wild - type ( A ) and OtofPga / Pga ( B ) ; stimulatory : wild - type ( C ) and OtofPga / Pga ( D ) . Tethered SVs are highlighted in orange with orange tether ( s ) ; single - tethers ( with orange arrowhead ) and multiple - tethers ( with brown arrowhead ) . The number of tethers per SV is indicated . Magnification : 12 , 000x ; scale bar , 40 nm . Virtual sections from inhibitory : wild - type ( E ) and OtofPga / Pga ( F ) ; stimulatory : wild - type ( G ) and OtofPga / Pga ( H ) . Docked SVs at the AZ - membrane ( blue line ) are highlighted ( orange asterisk ) . Some docked SVs are localized close to the presynaptic density ( PD , pink outline ) , anchoring the ribbon ( R ) . Magnification : 12 , 000x ; scale bar , 100 nm . Percentage of multiple - tethered MP - SVs per ribbon of the total MP - SV pool show more multiple - tethered SVs in OtofPga / Pga IHCs . Percentage of docked MP - SVs per ribbon of the total MP - pool . The number of docked SVs is significantly increased at OtofPga / Pga inhibited and specifically at stimulated AZs . P - values for ( I ) and ( J ) are calculated by Fisher ' s exact Chi - square test , wild - type : inhib , n = 102 SVs ; rest , n = 82 SVs ; stim , n = 99 SVs ; OtofPga / Pga : inhib , n = 123 SVs ; rest , n = 96 SVs ; stim , n = 111 SVs ; ns p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001Bar - plots show the distance of multiple - tethered ( O ) and docked ( P ) SVs from presynaptic density ( PD ) , 0 - 100 nm : MP - SV pool ( dark blue ) ; 100 - 500 nm : peripheral SV pool ( light blue ) . Upon stimulation , multiple - tethered and docked SVs are localized significantly closer to the PD in OtofPga / Pga . P - values for ( O ) and ( P ) are calculated by Wilcoxon Rank test , * p < 0 . 05 , * * * p < 0 . 001 , ns p > 0 . 05 and Ø inconclusive statistics because occurrence of docked SVs in wild - type resting condition is extremely rare . Multiple - tethered SVs , wild - type : inhib , n = 3 SVs ; rest , n = 13 SVs ; stim , n = 3 SVs ; OtofPga / Pga : inhib , n = 21 SVs ; rest , n = 34 SVs ; stim , n = 16 SVs ; docked SVs , wild - type : inhib , n = 2 SVs ; rest , n = 1 SVs ; stim , n = 7 SVs ; OtofPga / Pga : inhib , n = 20 SVs ; rest , n = 4 SVs ; stim , n = 18 SVs .

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 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 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

Figure 7Example tomographic virtual sections from inhibitory: wild-type (A) and OtofPga/Pga (B); stimulatory: wild-type (C) and OtofPga/Pga (D). Tethered SVs are highlighted in orange with orange tether(s); single-tethers (with orange arrowhead) and multiple-tethers (with brown arrowhead). The number of tethers per SV is indicated. Magnification: 12,000x; scale bar, 40 nm. Virtual sections from inhibitory: wild-type (E) and OtofPga/Pga (F); stimulatory: wild-type (G) and OtofPga/Pga (H). Docked SVs at the AZ-membrane (blue line) are highlighted (orange asterisk). Some docked SVs are localized close to the presynaptic density (PD, pink outline), anchoring the ribbon (R). Magnification: 12,000x; scale bar, 100 nm.Percentage of multiple-tethered MP-SVs per ribbon of the total MP-SV pool show more multiple-tethered SVs in OtofPga/Pga IHCs. Percentage of docked MP-SVs per ribbon of the total MP-pool. The number of docked SVs is significantly increased at OtofPga/Pga inhibited and specifically at stimulated AZs. P-values for (I) and (J) are calculated by Fisher's exact Chi-square test, wild-type: inhib, n = 102 SVs; rest, n = 82 SVs; stim, n = 99 SVs; OtofPga/Pga: inhib, n = 123 SVs; rest, n = 96 SVs; stim, n = 111 SVs; ns p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001Bar-plots show the distance of multiple-tethered (O) and docked (P) SVs from presynaptic density (PD), 0-100 nm: MP-SV pool (dark blue); 100-500 nm: peripheral SV pool (light blue). Upon stimulation, multiple-tethered and docked SVs are localized significantly closer to the PD in OtofPga/Pga. P-values for (O) and (P) are calculated by Wilcoxon Rank test, * p < 0.05, *** p < 0.001, ns p > 0.05 and Ø inconclusive statistics because occurrence of docked SVs in wild-type resting condition is extremely rare. Multiple-tethered SVs, wild-type: inhib, n = 3 SVs; rest, n = 13 SVs; stim, n = 3 SVs; OtofPga/Pga: inhib, n = 21 SVs; rest, n = 34 SVs; stim, n = 16 SVs; docked SVs, wild-type: inhib, n = 2 SVs; rest, n = 1 SVs; stim, n = 7 SVs; OtofPga/Pga: inhib, n = 20 SVs; rest, n = 4 SVs; stim, n = 18 SVs.


Figure 8Example virtual sections depict SVs with single membrane - ( orange arrowhead ) and presynaptic density ( pink arrowhead ) - attached tethers in resting wild - type . Magnification 12 , 000x ; scale bar , 40 nm . 3D reconstructions of same tethered SVs ( orange ) as shown in ( A ) and ( B ) ; membrane - attached ( A ' , yellow tether ) and presynaptic density ( PD ) - attached ( B ' , pink tether ) . Individual tether lengths are depicted . For clarity , ribbon ( R ) and AZ - membrane are shown in transparent . Scale bar , 20 nm . The graph depicts the mean length of single membrane - , presynaptic density - attached and multiple - tethers . Data are presented as mean ± SEM . Stimulated OtofPga / Pga synapses exhibit a shorter average tether length compared to stimulated wild - type . The cumulative probability distribution of MP - SVs plotted against the tether length . The red dotted lines represent the mean MP tether length in resting wild - type for each sub - pool . Total mean tether length in the MP - SV pool . Data are presented as mean ± SEM . Longer tethers are present upon stimulation in wild - type . Resting OtofPga / Pga ribbon synapses harbor the longest tethers , and tether where shorter at stimulated OtofPga / Pga synapses compared to wild - type . The red dotted lines represent the mean MP tether length in resting wild - type for each sub - pool .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8Example virtual sections depict SVs with single membrane- (orange arrowhead) and presynaptic density (pink arrowhead) -attached tethers in resting wild-type. Magnification 12,000x; scale bar, 40 nm. 3D reconstructions of same tethered SVs (orange) as shown in (A) and (B); membrane-attached (A', yellow tether) and presynaptic density (PD) -attached (B', pink tether). Individual tether lengths are depicted. For clarity, ribbon (R) and AZ-membrane are shown in transparent. Scale bar, 20 nm.The graph depicts the mean length of single membrane-, presynaptic density-attached and multiple-tethers. Data are presented as mean ± SEM. Stimulated OtofPga/Pga synapses exhibit a shorter average tether length compared to stimulated wild-type.The cumulative probability distribution of MP-SVs plotted against the tether length. The red dotted lines represent the mean MP tether length in resting wild-type for each sub-pool.Total mean tether length in the MP-SV pool. Data are presented as mean ± SEM. Longer tethers are present upon stimulation in wild-type. Resting OtofPga/Pga ribbon synapses harbor the longest tethers, and tether where shorter at stimulated OtofPga/Pga synapses compared to wild-type. The red dotted lines represent the mean MP tether length in resting wild-type for each sub-pool.


Figure 1One - cell stage embryos were injected animally with mRNA encoding dkk1 ( 35 / 45 pg ) protein . Total RNA was isolated from ten control embryos at neurula stage 17 ( lane 2 ) and ten embryos from each dkk1 injected group ( lanes 3 - 4 ) . Various neural AP markers were examined by sqRT - PCR : cement gland and forebrain ( xag1 , xanf1 , otx2 ) , hindbrain ( krox20 , hoxb3 ) , hindbrain / spinal cord border ( hoxb4 ) , and spinal cord ( hoxb9 , cdx1 - 2 - 4 , hoxd10 ) . Ef1α serves as a control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) . One - cell stage embryos were injected animally with the zygotic - expressing Nxfz8 / pCS2 encoding plasmid ( 100 / 200 pg ) . Total RNA was isolated from ten control embryos at neurula stage 17 ( lane 2 ) and ten embryos from each Nxfz8 injected group ( lanes 3 - 4 ) . Various neural AP markers were examined by sqRT - PCR : cement gland and forebrain ( xag1 , xanf1 , otx2 ) , hindbrain ( krox20 , hoxb3 ) , hindbrain / spinal cord border ( hoxb4 ) , and spinal cord ( hoxb9 , cdx1 - 2 - 4 , hoxd10 ) . Ef1α serves as a control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) . Embryos from the same experiment as in ( A ) underwent in situ hybridization . Expression of the hindbrain marker krox20 ( upper two panels ) was reduced by Dkk1 ( 45pg ) in 83 % of the embryos ( n = 18 ) . Expression of spinal cord markers ( hoxb9 , cdx4 , hox10 ) was normal , being unchanged in 89 % of the embryos ( n = 18 for each marker ) . Control embryos , ( CE ) .

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 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 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

Figure 1One-cell stage embryos were injected animally with mRNA encoding dkk1 (35/45 pg) protein. Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each dkk1 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).One-cell stage embryos were injected animally with the zygotic-expressing Nxfz8/pCS2 encoding plasmid (100/200 pg). Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each Nxfz8 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).Embryos from the same experiment as in (A) underwent in situ hybridization. Expression of the hindbrain marker krox20 (upper two panels) was reduced by Dkk1 (45pg) in 83% of the embryos (n=18). Expression of spinal cord markers (hoxb9, cdx4, hox10) was normal, being unchanged in 89% of the embryos (n=18 for each marker). Control embryos, (CE).


Figure 2One - cell stage embryos were injected animally with the Zic1 - MO ( 25 / 40 ng ) . Total RNA was isolated from ten control embryos at neurula stage 18 ( lane 2 ) and ten embryos from each Zic1 - MO injected group ( lanes 3 - 4 ) . Neural AP markers were examined by sqRT - PCR : forebrain ( xanf1 , otx2 ) , hindbrain ( krox20 , hoxb1 , hoxb3 ) , spinal cord ( hoxb9 , cdx1 - 4 ) and panneural ( soxd ) . Ef1α is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) . One - cell stage embryos were injected animally with mRNA encoding Zic5 dominant - negative ( 0 . 5 ng ) protein . Total RNA was isolated from five control embryos at neurula stage 18 ( lane 1 ) and five embryos from the injected group ( lanes 2 ) . Neural AP markers were examined by sqRT - PCR : hindbrain ( krox20 ) and spinal cord ( cdx1 - 4 ) . Ef1α is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 3 ) . One - cell stage embryos were injected animally with the Zic1 - MO ( 25 ng ) . Embryos underwent in situ hybridization . Expression of the panneural sox2 marker was normal , the forebrain marker xanf1 was expanded in 80 % of the embryos , the hindbrain representative marker krox20 was reduced in 80 % of the embryos and the representative spinal cord marker cdx4 was expanded in 90 % of the embryos . N = 23 embryos were per marker .

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 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

Figure 2One-cell stage embryos were injected animally with the Zic1-MO (25/40 ng). Total RNA was isolated from ten control embryos at neurula stage 18 (lane 2) and ten embryos from each Zic1-MO injected group (lanes 3-4). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).One-cell stage embryos were injected animally with mRNA encoding Zic5 dominant-negative (0.5 ng) protein. Total RNA was isolated from five control embryos at neurula stage 18 (lane 1) and five embryos from the injected group (lanes 2). Neural AP markers were examined by sqRT-PCR: hindbrain (krox20) and spinal cord (cdx1-4). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 3).One-cell stage embryos were injected animally with the Zic1-MO (25 ng). Embryos underwent in situ hybridization. Expression of the panneural sox2 marker was normal, the forebrain marker xanf1 was expanded in 80% of the embryos, the hindbrain representative marker krox20 was reduced in 80% of the embryos and the representative spinal cord marker cdx4 was expanded in 90% of the embryos. N = 23 embryos were per marker.


Figure 3One - cell stage embryos were injected animally with mRNAs encoding the noggin ( 5 / 20 pg ) and / or meis3 ( 0 . 5 ng ) proteins . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 17 . Total RNA was isolated from seven control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 8 ) . Neural AP markers were examined by sqRT - PCR : forebrain ( xanf1 , otx2 ) , hindbrain ( krox20 , hoxb1 , hoxb3 ) , spinal cord ( hoxb9 , cdx1 - 4 ) and panneural ( soxd ) . Ef1α is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) . One - cell stage embryos were injected animally with mRNA encoding bmp4 ( 0 . 2 ng ) protein . Total RNA was isolated from five control embryos at neurula stage 17 ( lane 1 ) and five embryos from the injected group ( lanes 2 ) . Neural and mesodermal markers were examined by sqRT - PCR : forebrain ( otx2 ) , hindbrain ( krox20 , hoxb3 ) , spinal cord ( hoxb9 , hoxc10 , cdx4 ) , panneural ( nrp1 , ncam ) and mesodermal ( muscle actin - MA ) . ODC is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) . One - cell stage embryos were injected animally with mRNA encoding bmp4 ( 0 . 2 ng ) protein Embryos underwent in situ hybridization . Expression of the hindbrain marker hoxb3 was reduced / eliminated by bmp4 ( 0 . 2 ng ) in 68 % of the embryos ( n = 19 ) . Expression of the spinal cord marker cdx4 was slightly increased / normal , in 100 % of the embryos ( n = 19 ) .

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Figure 3One-cell stage embryos were injected animally with mRNAs encoding the noggin (5/20 pg) and/or meis3 (0.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 17. Total RNA was isolated from seven control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein. Total RNA was isolated from five control embryos at neurula stage 17 (lane 1) and five embryos from the injected group (lanes 2). Neural and mesodermal markers were examined by sqRT-PCR: forebrain (otx2), hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), panneural (nrp1, ncam) and mesodermal (muscle actin - MA). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein Embryos underwent in situ hybridization. Expression of the hindbrain marker hoxb3 was reduced/eliminated by bmp4 (0.2 ng) in 68% of the embryos (n=19). Expression of the spinal cord marker cdx4 was slightly increased/normal, in 100% of the embryos (n=19).


Figure 4One - cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA ( 5 - 50 pg ) . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 16 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 7 ) . Neural , epidermal and mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 ) , spinal cord ( hoxb9 , hoxc10 / d10 , cdx1 - 2 - 4 ) , epidermal cytokeratin ( E13 ) , lateral mesoderm ( osr1 - 2 ) and dorsal - lateral mesoderm muscle actin ( MA ) . ODC is the control for quantitating the RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) . One - cell stage embryos were injected animally with mRNAs encoding the bmp4 ( 50 / 150 pg ) and / or meis3 ( 0 . 7 ng ) proteins . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 16 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 8 ) . Neural and mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 ) , spinal cord ( hoxb9 , hoxc10 , cdx2 ) , and lateral mesoderm ( osr1 ) . Ef1α is the control for quantitating the RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) . One - cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA ( 75 - 200 pg ) . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to gastrula stage 11 - 11 . 5 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 6 ) . Marker expression was determined by sqRT - PCR . Xenopus brachyury ( xbra ) expression was induced by BMP4 in a dose - dependent manner . Marker expression was determined by sqRT - PCR . Neither dorsal ( chd , goosecoid - gsc ) or ventral - lateral ( osr1 ) mesoderm markers were induced by BMP4 . Sox2 / 3 neural markers are expressed in the ACs and are unchanged by BMP expression . EF1α is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-7). Neural, epidermal and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10/d10, cdx1-2-4), epidermal cytokeratin (E13), lateral mesoderm (osr1-2) and dorsal-lateral mesoderm muscle actin (MA). ODC is the control for quantitating the RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50/150 pg) and/or meis3 (0.7 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10, cdx2), and lateral mesoderm (osr1). Ef1α is the control for quantitating the RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (75-200 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to gastrula stage 11-11.5. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). Marker expression was determined by sqRT-PCR. Xenopus brachyury (xbra) expression was induced by BMP4 in a dose-dependent manner. Marker expression was determined by sqRT-PCR. Neither dorsal (chd, goosecoid-gsc) or ventral-lateral (osr1) mesoderm markers were induced by BMP4. Sox2/3 neural markers are expressed in the ACs and are unchanged by BMP expression. EF1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


Figure 5One - cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA ( 5 - 50 pg ) . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 16 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 6 ) . FGF3 - 4 - 8 expression was determined by sqRT - PCR . ODC is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) . One - cell stage embryos were injected animally with mRNAs encoding the bmp4 ( 50 pg ) and / or FGF dominant - negative receptor ( FGF - DNR , 1 . 5 ng ) proteins . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 16 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 6 ) . In addition to fgf3 , neural , epidermal and mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 , hoxb3 ) , spinal cord ( hoxb9 , hoxc10 , cdx4 ) , lateral mesoderm ( osr1 ) , dorsal - lateral mesoderm muscle actin ( MA ) , and epidermal cytokeratin ( E13 ) . ODC is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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Figure 5One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). FGF3-4-8 expression was determined by sqRT-PCR. ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50 pg) and/or FGF dominant-negative receptor (FGF-DNR, 1.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). In addition to fgf3, neural, epidermal and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), lateral mesoderm (osr1), dorsal-lateral mesoderm muscle actin (MA), and epidermal cytokeratin (E13). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


Figure 1A . Representative immunoblots and quantifications ( below ) of the indicated proteins in lysates of ARPE - 19 cells treated with 1 mM DFP for the indicated time ( h , hours ) . B - C . Representative confocal images of ARPE - 19 cells stably expressing the mito - QC reporter ( B ) , or the pexo - QC reporter ( C ) treated with 1 mM DFP for the indicated time ( h , hours ) . Enlarged images of the area outlined in white are shown in the lower corners . The ' red - only ' mask is generated during images analysis and highlights red - only puncta ( autolysosomes ) based on the mCherry / GFP ratio and thresholding criteria . Nuclei were stained in blue ( Hoechst ) . Scale bar : 10μm . At right , quantification of total red - only punctate per cell ( mitolysosomes or pexolysosomes ) . D . ARPE - 19 cells were transfected with 50 pmol of non - targeting siRNA ( siNT ) or 50 pmol of siRNA targeting ATG13 or ULK1 . 48 hours post knockdown , cells were treated with 1 mM DFP for an additional 48 hours . Cell lysates were subject to immunoblotting with the indicated antibodies . Quantification is shown on the right .

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Figure 1A. Representative immunoblots and quantifications (below) of the indicated proteins in lysates of ARPE-19 cells treated with 1 mM DFP for the indicated time (h, hours).B-C. Representative confocal images of ARPE-19 cells stably expressing the mito-QC reporter (B), or the pexo-QC reporter (C) treated with 1 mM DFP for the indicated time (h, hours). Enlarged images of the area outlined in white are shown in the lower corners. The 'red-only' mask is generated during images analysis and highlights red-only puncta (autolysosomes) based on the mCherry/GFP ratio and thresholding criteria. Nuclei were stained in blue (Hoechst). Scale bar: 10μm. At right, quantification of total red-only punctate per cell (mitolysosomes or pexolysosomes).D. ARPE-19 cells were transfected with 50 pmol of non-targeting siRNA (siNT) or 50 pmol of siRNA targeting ATG13 or ULK1. 48 hours post knockdown, cells were treated with 1 mM DFP for an additional 48 hours. Cell lysates were subject to immunoblotting with the indicated antibodies. Quantification is shown on the right.


Figure 2A . Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells , treated with 1mM DFP for 48 hours . B . Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo - QC reporter and treated with 1 mM DFP for 48 hours ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) . C . Representative immunoblots ( left panel ) and quantification ( right panel ) of the indicated proteins in lysates of WT or two NIX KO ARPE - 19 clones ( Cl ) , treated with 1 mM DFP for the indicated time ( h , hours ) . D - E . Representative confocal images of WT or NIX KO ARPE - 19 cells stably expressing the mito - QC reporter ( D ) or the pexo - QC reporter ( E ) treated with 1 mM DFP for 48 hours ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) . F . Representative immunoblots ( left panel ) and quantification ( right panel ) of the indicated proteins . NIX KO ARPE - 19 cells ( Cl 31 ) stably expressing a pBabe empty vector ( ∅ ) or a pBabe NIX vector , were treated with 1 mM DFP for 48 hours prior lysis . Two biological replicates are shown in the immunoblot .

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

Figure 2A. Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells, treated with 1mM DFP for 48 hours.B. Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo-QC reporter and treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).C. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins in lysates of WT or two NIX KO ARPE-19 clones (Cl), treated with 1 mM DFP for the indicated time (h, hours).D-E. Representative confocal images of WT or NIX KO ARPE-19 cells stably expressing the mito-QC reporter (D) or the pexo-QC reporter (E) treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).F. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins. NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe empty vector (∅) or a pBabe NIX vector, were treated with 1 mM DFP for 48 hours prior lysis. Two biological replicates are shown in the immunoblot.


Figure 3A - B . Representative confocal images of untreated ( CTRL ) and DFP - treated ARPE - 19 cells ( 1mM , 48 hours ) , stably expressing GFP - WT NIX ( green ) and stained with anti - catalase antibody ( A , red ) or anti - PMP70 antibody ( B , red ) . At right , quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70 , with or without DFP treatment for 48 hours . Peroxisomes enriched for GFP - NIX or having minimal signal for GFP - NIX are depicted with white arrowheads or white arrows , respectively . C . Representative confocal images of ARPE - 19 cells stably expressing GFP - WT NIX , GFP - NIXΔTM or GFP - NIX TM only ( green ) , treated with DFP ( 1mM ) for 48 hours and immunostained with anti - catalase antibody ( red ) . At right , quantification of Pearson correlation coefficients between the green and the red signal .

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

Figure 3A-B. Representative confocal images of untreated (CTRL) and DFP-treated ARPE-19 cells (1mM, 48 hours), stably expressing GFP-WT NIX (green) and stained with anti-catalase antibody (A, red) or anti-PMP70 antibody (B, red). At right, quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70, with or without DFP treatment for 48 hours. Peroxisomes enriched for GFP-NIX or having minimal signal for GFP-NIX are depicted with white arrowheads or white arrows, respectively.C. Representative confocal images of ARPE-19 cells stably expressing GFP-WT NIX, GFP-NIXΔTM or GFP-NIX TM only (green), treated with DFP (1mM) for 48 hours and immunostained with anti-catalase antibody (red). At right, quantification of Pearson correlation coefficients between the green and the red signal.


Figure 4B . Representative immunoblots of the indicated proteins in lysates of WT or HA - Parkin ( PRKN ) - ARPE - 19 cells treated as in A .

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Figure 4B. Representative immunoblots of the indicated proteins in lysates of WT or HA-Parkin(PRKN)- ARPE-19 cells treated as in A.


Figure 5B . Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE - 19 cells ( Cl 31 ) stably expressing a pBabe Flag vector ( ∅ ) , a pBabe Flag - NIX vector ( NIX ) , a pBabe Flag - NIX W36A \ L39A vector ( W36A \ L39A ) or a pBabe Flag - NIX S34E \ S35E vector ( S34E \ S35E ) , and treated with 1 mM DFP for 48 hours prior lysis . C - D . Representative confocal images of cells as in B , stably expressing the mito - QC reporter ( C ) or the pexo - QC reporter ( D ) and treated with 1 mM DFP for 24 hours ( C ) or 48 hours ( D ) ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) . Enlarged images of the area outlined in white are shown in the lower corners . Nuclei were stained in blue ( Hoechst )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B. Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe Flag vector (∅), a pBabe Flag-NIX vector (NIX), a pBabe Flag-NIX W36A\L39A vector (W36A\L39A) or a pBabe Flag-NIX S34E\S35E vector (S34E\S35E), and treated with 1 mM DFP for 48 hours prior lysis.C-D. Representative confocal images of cells as in B, stably expressing the mito-QC reporter (C) or the pexo-QC reporter (D) and treated with 1 mM DFP for 24 hours (C) or 48 hours (D) (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel). Enlarged images of the area outlined in white are shown in the lower corners. Nuclei were stained in blue (Hoechst)


Figure 6A . Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers ( PMP70 and catalase ) or a lysosomal marker LAMP1 . Nuclei are coloured in white ( DAPI ) . RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL ; inner nuclear layer ; IPL , inner plexiform layer ; RGC , retinal ganglion cells . B . Quantification of signal area in the ONL and INL from images as A . Each data point represents a single mouse ( n = 3 - 4 per group ) .

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Figure 6A. Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers (PMP70 and catalase) or a lysosomal marker LAMP1. Nuclei are coloured in white (DAPI). RPE, retinal pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL; inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cells. B. Quantification of signal area in the ONL and INL from images as A. Each data point represents a single mouse (n=3-4 per group).


Figure 2GST - Mif2 _ 1 - 41 was immobilized on GSH Sepharose beads and incubated with Mtw1 - Nnf1 complex ( MN ) in the presence of increasing concentrations of Ame1 _ 1 - 20 peptide ( lanes 4 - 9 ) . GST - Ame1 _ 1 - 30 served as control ( lane 2 and 3 ) . After washing , bound complexes were eluted by adding SDS sample buffer . Protein bands in Coomassie stained gel are highlighted in the indicated color . Integrated density of Mtw1 bands was quantified using ImageJ and correlated to GST - Mif2 amounts in the same lane . Binding of FITC - Mif21 - 41 to MN was directly determined in a fluorescence polarization ( FP ) assay . This interaction at EC50 was than competed with increasing concentration of Ame11 - 20 unlabeled peptide . Both experiments were performed in triplicates of which the mean and standard deviation is shown . Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to the MN complex . Mif2 and Ame1 wild - type N - termini serve as a control , protein bands are highlighted in the indicated color .

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Figure 2GST-Mif2_1-41 was immobilized on GSH Sepharose beads and incubated with Mtw1-Nnf1 complex (MN) in the presence of increasing concentrations of Ame1_1-20 peptide (lanes 4-9). GST-Ame1_1-30 served as control (lane 2 and 3). After washing, bound complexes were eluted by adding SDS sample buffer. Protein bands in Coomassie stained gel are highlighted in the indicated color. Integrated density of Mtw1 bands was quantified using ImageJ and correlated to GST-Mif2 amounts in the same lane.Binding of FITC-Mif21-41 to MN was directly determined in a fluorescence polarization (FP) assay. This interaction at EC50 was than competed with increasing concentration of Ame11-20 unlabeled peptide. Both experiments were performed in triplicates of which the mean and standard deviation is shown.Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to the MN complex. Mif2 and Ame1 wild-type N-termini serve as a control, protein bands are highlighted in the indicated color.


Figure 3Analysis of these Mtw1 mutants in vivo using the anchor - away technique . Mtw1 - FRB strains containing RPL13 - FKBP12 for cytoplasmic anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 ° C . Tetrad analysis of Mtw1 - wt or mutants . Surviving spores in which the Mtw1 - wt or mutant rescue construct was the only source of Mtw1 are indicated in pink circles . Western blotting confirming the expression of Mtw1 - 6x - Flag wt or mutant proteins in the Mtw1 - FRB strain background . Analytical SEC runs and accompanying SDS - PAGE of Mtw1c - wt or - 5A mutant ( blue ) , AO ( grey ) or a stoichiometric combination of both complexes ( green ) at 10 µM each . Boxes indicate corresponding fractions in left and right panel . Note that the same Ame1 - Okp1 elution profile and SDS - PAGE ( middle panel ) is displayed in both sets to improve clarity .

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Figure 3Analysis of these Mtw1 mutants in vivo using the anchor-away technique. Mtw1-FRB strains containing RPL13-FKBP12 for cytoplasmic anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.Tetrad analysis of Mtw1-wt or mutants. Surviving spores in which the Mtw1-wt or mutant rescue construct was the only source of Mtw1 are indicated in pink circles.Western blotting confirming the expression of Mtw1-6x-Flag wt or mutant proteins in the Mtw1-FRB strain background.Analytical SEC runs and accompanying SDS-PAGE of Mtw1c-wt or -5A mutant (blue), AO (grey) or a stoichiometric combination of both complexes (green) at 10 µM each. Boxes indicate corresponding fractions in left and right panel. Note that the same Ame1-Okp1 elution profile and SDS-PAGE (middle panel) is displayed in both sets to improve clarity.


Figure 4Analysis of Ame1 N - terminal deletion and swap mutant using the anchor - away technique . Ame1 - FRB strains containing RPL13 - FKBP12 for ribosome anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 ° C . Expression levels of 3xHA tagged Ame1 rescue constructs from ( B ) analyzed by western blotting . Tetrad dissection of strains where one endogenous copy of Ame1 was replaced with the indicated construct . Phenotypic analysis of different Ame1 rescue constructs using fluorescence microscopy . Images of representative large budded cells immediately and 4 h after Rapamycin addition are shown , indicated as t = 0 h and t = 4 h . Brightfield images show the morphology of the corresponding yeast cell . Only large budded cells were included in the analysis . One kinetochore cluster is enlarged in the white box . Scale bar represents 5 µm . Quantification of ( E ) in which large budded cells were classified according to their Mtw1 - GFP signal into the three indicated groups . Scale bar represents 5 µm . A total of 100 cells was quantified for every strain . Mean values of the three indicated groups were calculated and plotted in a bar chart . Error bars indicate the SEM of 3 independent experiments . Analysis of Mif2 N - terminal and swap mutant using the anchor - away technique as for ( B ) . Phenotypes of the indicated Mif2 constructs were observed in an otherwise wild - type or ctf19 ∆ background . Western blotting showing 6xFlag tagged Mif2 expression levels of strains from ( G ) .

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Figure 4Analysis of Ame1 N-terminal deletion and swap mutant using the anchor-away technique. Ame1-FRB strains containing RPL13-FKBP12 for ribosome anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.Expression levels of 3xHA tagged Ame1 rescue constructs from (B) analyzed by western blotting.Tetrad dissection of strains where one endogenous copy of Ame1 was replaced with the indicated construct.Phenotypic analysis of different Ame1 rescue constructs using fluorescence microscopy. Images of representative large budded cells immediately and 4 h after Rapamycin addition are shown, indicated as t = 0 h and t = 4 h. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. One kinetochore cluster is enlarged in the white box. Scale bar represents 5 µm.Quantification of (E) in which large budded cells were classified according to their Mtw1-GFP signal into the three indicated groups. Scale bar represents 5 µm. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 3 independent experiments.Analysis of Mif2 N-terminal and swap mutant using the anchor-away technique as for (B). Phenotypes of the indicated Mif2 constructs were observed in an otherwise wild-type or ctf19∆ background.Western blotting showing 6xFlag tagged Mif2 expression levels of strains from (G).


Figure 5Co - purification of Strep - Mtw1c through Flag - Mif2 after co - expression in insect cells . Proteins were detected by western blotting using their respective tags . Western blotting of elution fractions from an SEC experiment of Mif2 - wt or Mif2Ame1 - N swap in the absence ( upper panel ) or presence ( lower panel ) of Mtw1c . ITC analysis of Mif21 - 41 or Ame11 - 20 binding to MN . Experiments were performed 3 or 4 times , respectively . One representative binding curve is shown , whereas the KD value represents the average of all experiments . Cross - link ( XL ) MS analysis of the Mif2wt protein in isolation or bound to Cse4n using the cross - linker Bis [ sulfosuccinimidyl ] glutarate . Mif2wt alone or in combination with Cse4n were subjected to analytical size exclusion chromatography at 10 µM concentration . The corresponding Coomassie - stained SDS - PAGE gels of Mif2 - wt alone or in combination with Cse4n are shown . Fractions used for cross - linking are highlighted . Topological map of Mif2 based on the identified intra - molecular cross - links . Common cross links that are found in both samples are shown in red , whereas unique crosslinks that can be only found in one of the samples are highlighted in blue . Conserved regions and their described functions are indicated . A list of cross - links that link N - terminal residues to C - terminal regions of Mif2 is shown for Mif2 alone and bound to Cse4n leading to a proposed conformation of Mif2 .

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Figure 5Co-purification of Strep-Mtw1c through Flag-Mif2 after co-expression in insect cells. Proteins were detected by western blotting using their respective tags.Western blotting of elution fractions from an SEC experiment of Mif2-wt or Mif2Ame1-N swap in the absence (upper panel) or presence (lower panel) of Mtw1c.ITC analysis of Mif21-41 or Ame11-20 binding to MN. Experiments were performed 3 or 4 times, respectively. One representative binding curve is shown, whereas the KD value represents the average of all experiments.Cross-link (XL) MS analysis of the Mif2wt protein in isolation or bound to Cse4n using the cross-linker Bis[sulfosuccinimidyl] glutarate. Mif2wt alone or in combination with Cse4n were subjected to analytical size exclusion chromatography at 10 µM concentration. The corresponding Coomassie-stained SDS-PAGE gels of Mif2-wt alone or in combination with Cse4n are shown. Fractions used for cross-linking are highlighted. Topological map of Mif2 based on the identified intra-molecular cross-links. Common cross links that are found in both samples are shown in red, whereas unique crosslinks that can be only found in one of the samples are highlighted in blue. Conserved regions and their described functions are indicated. A list of cross-links that link N-terminal residues to C-terminal regions of Mif2 is shown for Mif2 alone and bound to Cse4n leading to a proposed conformation of Mif2.


Figure 6Mif2Flag - wt or - ∆ sigAT directly from insect cell lysate was immobilized on M2 Flag Agarose , washed and incubated with Mtw1 - Nnf1 complex ( MN ) . After washing , bound complexes were eluted by adding 3xFlag peptide . Western blotting against the His - tag on Nnf1 confirmed increased binding of MN to Mif2 - ∆ sigAT , already visible in the Coomassie stained SDS - PAGE gel . Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mif2 - wt or Mif2 - ∆ sigAT and MN alone and in combination at 10 µM concentration . All combinations were incubated for 1 h at 4 ° C prior to the run . MN ( blue ) , Mif2 ( red ) , combination ( green ) . Note that the same MN elution profile and SDS - PAGE ( upper panel ) is displayed in both sets to improve clarity . Dashed boxes highlighting corresponding fractions were included to improve comparability .

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Figure 6Mif2Flag-wt or -∆sigAT directly from insect cell lysate was immobilized on M2 Flag Agarose, washed and incubated with Mtw1-Nnf1 complex (MN). After washing, bound complexes were eluted by adding 3xFlag peptide. Western blotting against the His-tag on Nnf1 confirmed increased binding of MN to Mif2-∆sigAT, already visible in the Coomassie stained SDS-PAGE gel.Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mif2-wt or Mif2-∆sigAT and MN alone and in combination at 10 µM concentration. All combinations were incubated for 1 h at 4°C prior to the run. MN (blue), Mif2 (red), combination (green). Note that the same MN elution profile and SDS-PAGE (upper panel) is displayed in both sets to improve clarity. Dashed boxes highlighting corresponding fractions were included to improve comparability.


Figure 7Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Cse4n ( light blue ) , Mif2 - wt ( red ) , combination ( green ) . Note that the same Cse4n ( upper panel ) and Mif2 ( middle panel ) elution profiles and SDS - PAGEs are also displayed as reference in B and C ( Mif2 ) or C ( Cse4n ) to improve clarity . Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Mif2 - wt ( red ) , combination ( green ) . Note that the same Mtw1c ( upper panel ) elution profile and SDS - PAGE is also displayed as reference in C to improve clarity . Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Cse4n ( light blue ) , Mif2 - wt ( red ) , combination ( green ) . Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Cse4n ( light blue ) , Mif2Ame - 1N swap ( red ) , combination ( green ) . Note that the same Cse4n ( upper panel ) and Mif2Ame - 1N swap ( middle panel ) elution profiles and SDS - PAGEs are also displayed as reference in E and F ( Mif2Ame - 1N swap ) or F ( Cse4n ) to improve clarity . Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Mif2Ame - 1N swap ( red ) , combination ( green ) . Note that the same Mtw1c ( upper panel ) elution profile and SDS - PAGE is also displayed as reference in F to improve clarity . Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Cse4n ( light blue ) , Mif2Ame - 1N swap ( red ) , combination ( green ) .

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Figure 7Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Cse4n (light blue), Mif2-wt (red), combination (green). Note that the same Cse4n (upper panel) and Mif2 (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in B and C (Mif2) or C (Cse4n) to improve clarity.Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Mif2-wt (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in C to improve clarity.Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Cse4n (light blue), Mif2-wt (red), combination (green).Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Cse4n (light blue), Mif2Ame-1N swap (red), combination (green). Note that the same Cse4n (upper panel) and Mif2Ame-1N swap (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in E and F (Mif2Ame-1N swap) or F (Cse4n) to improve clarity.Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Mif2Ame-1N swap (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in F to improve clarity.Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Cse4n (light blue), Mif2Ame-1N swap (red), combination (green).


Figure 8Phenotypic analysis of Mif2 mutants using a galactose inducible overexpression system . Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS - Glucose or doHIS - Raffinose / Galactose plates and incubated at 30 ° C . Serial dilution assay of chromosomally integrated Mif2 variants under the control of the pGAL promoter . Strains were plated on YEP - Glucose and YEP - Raffinose / Galactose plates . Analysis of chromosome segregation in strains from ( B ) . Distribution of fluorescently labeled Chromosome III was analyzed in large - budded cells 16 hours after switching the medium to Galactose . Scale bar represents 2 μm . Microscopic analysis of kinetochores in Mif2 overexpressing cells visualizing Mtw1 - GFP and Spc42 - mCherry . Images of representative large budded cells after 16h in Galactose are shown . One kinetochore cluster is enlarged in the white box for each strain . Scale bar represents 5 µm . Brightfield images show the morphology of the corresponding yeast cell . Only large budded cells were included in the analysis . Quantification of ( D ) in which large budded cells were classified according to their Mtw1 - GFP signal into the four indicated groups . A total of 100 cells was quantified for every strain . Mean values of the three indicated groups were calculated and plotted in a bar chart . Error bars indicate the SEM of 2 independent experiments . Co - IP experiment of indicated Mif2 - Flag variants and Mtw1 - Myc from yeast cell extracts . Phenotypic analysis of Mif2 - ∆ sigAT mutant using the same galactose inducible overexpression system as in ( A ) . Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS - Glucose or doHIS - Raffinose / Galactose plates and incubated at 30 ° C .

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Figure 8Phenotypic analysis of Mif2 mutants using a galactose inducible overexpression system. Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.Serial dilution assay of chromosomally integrated Mif2 variants under the control of the pGAL promoter. Strains were plated on YEP-Glucose and YEP-Raffinose/Galactose plates.Analysis of chromosome segregation in strains from (B). Distribution of fluorescently labeled Chromosome III was analyzed in large-budded cells 16 hours after switching the medium to Galactose. Scale bar represents 2 μm.Microscopic analysis of kinetochores in Mif2 overexpressing cells visualizing Mtw1-GFP and Spc42-mCherry. Images of representative large budded cells after 16h in Galactose are shown. One kinetochore cluster is enlarged in the white box for each strain. Scale bar represents 5 µm. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. Quantification of (D) in which large budded cells were classified according to their Mtw1-GFP signal into the four indicated groups. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 2 independent experiments.Co-IP experiment of indicated Mif2-Flag variants and Mtw1-Myc from yeast cell extracts.Phenotypic analysis of Mif2-∆sigAT mutant using the same galactose inducible overexpression system as in (A). Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.


Figure 1 . ( A and B ) Immunoblot of 9 - mo - old quadricepsmuscle lysates from control ( cont ) or VCP - WT , - RH9 , or - RH12 mutant transgenic lines with p62 ( A ) or LC3 ( B ) antibodies . Note the increase in p62 and LC3II isoforms in mutant animals . Densitometric quantification is from more than six 9 - mo - old animals / group . LC3 and p62 levels are normalized to loading control . Error bars represent the standard error from six independent experiments . * * , P < 0 . 001 . AU , arbitrary unit . ( C ) p62 immunostaining of tibialis anterior ( TA ) or quadriceps ( Quad ) muscle from 9 - mo - old control , VCP - WT , or one of two VCP - RH transgenic lines ( RH9 or RH12 ) . Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions . ( D ) Histochemistry and immunohistochemistry of quadriceps muscle from VCP - RH - expressing transgenic mice . Hematoxylin and eosin ( H & amp ; amp ; E ) staining of a 15 - mo - old animal with an RV , p62 immunostaining of an RV from a 12 - mo - old animal , LC3 immunostaining of an RV , and subsarcolemmal accumulations of LC3 from a 12 - mo - old animal . The bracket highlights one LC3 - positive myofiber . A single muscle fiber is outlined in white . ( C and D ) Arrows denote vacuoles or accumulations of p62 or LC3 . Bars , 30 µm .

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Figure 1.(A and B) Immunoblot of 9-mo-old quadricepsmuscle lysates from control (cont) or VCP-WT, -RH9, or -RH12 mutant transgenic lines with p62 (A) or LC3 (B) antibodies. Note the increase in p62 and LC3II isoforms in mutant animals. Densitometric quantification is from more than six 9-mo-old animals/group. LC3 and p62 levels are normalized to loading control. Error bars represent the standard error from six independent experiments. **, P < 0.001. AU, arbitrary unit.(C) p62 immunostaining of tibialis anterior (TA) or quadriceps (Quad) muscle from 9-mo-old control, VCP-WT, or one of two VCP-RH transgenic lines (RH9 or RH12). Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions.(D) Histochemistry and immunohistochemistry of quadriceps muscle from VCP-RH-expressing transgenic mice. Hematoxylin and eosin (H&amp;E) staining of a 15-mo-old animal with an RV, p62 immunostaining of an RV from a 12-mo-old animal, LC3 immunostaining of an RV, and subsarcolemmal accumulations of LC3 from a 12-mo-old animal. The bracket highlights one LC3-positive myofiber. A single muscle fiber is outlined in white. (C and D) Arrows denote vacuoles or accumulations of p62 or LC3. Bars, 30 µm.


Figure 2 . ( A ) Lysates from scrambled siRNA or VCP - targeted siRNA - treated U20S cells immunoblotted with antibodies to VCP , LC3 , p62 , or α - tubulin . Densitometric analysis is graphically represented from four independent experiments . LC3 and p62 levels are normalized to loading control . ( B ) Fluorescent microscopy images of siRNA scrambled control ( Scr ) or VCP - targeted siRNA KD cells expressing GFP - LC3 . Basal numbers of GFP - LC3 puncta / cell were counted . ( C ) Lysates from control or myc - tagged VCP - WT , ATPase - inactive VCP - EQ , or one of two IBMPFD mutants , VCP - RH or - AE , expressed for 16 h from stably transfected tetracycline - inducible U20S cells and immunoblotted with antibodies to myc , LC3 , p62 , or α - tubulin . Densitometric analysis is graphically represented from four independent experiments . LC3 and p62 levels are normalized to loading control . Note the increase in LC3II and p62 in VCP siRNA KD and mutant - expressing cells . ( D ) Fluorescent microscopy images of tetracycline - inducible U20S cells expressing GFP - LC3 treated with and without Baf for 4 h ( DMSO control [ left ] or Baf + [ right ] ) . Cells are control U20S cells and VCP - WT , - RH , - AE , and - EQ . ( E ) The number of GFP - LC3 puncta / cell was counted for control U20S and VCP - WT , - EQ , - RH , and - AE with and without Baf for 4 h ( DMSO or Baf ) . Note the increase in basal GFP - LC3 puncta in mutant - expressing cells . ( A - C and E ) Error bars represent the standard error from four independent experiments . * , P < 0 . 01 ; and * * , P < 0 . 001 . Bars , 25 µm .

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Figure 2.(A) Lysates from scrambled siRNA or VCP-targeted siRNA-treated U20S cells immunoblotted with antibodies to VCP, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control.(B) Fluorescent microscopy images of siRNA scrambled control (Scr) or VCP-targeted siRNA KD cells expressing GFP-LC3. Basal numbers of GFP-LC3 puncta/cell were counted.(C) Lysates from control or myc-tagged VCP-WT, ATPase-inactive VCP-EQ, or one of two IBMPFD mutants, VCP-RH or -AE, expressed for 16 h from stably transfected tetracycline-inducible U20S cells and immunoblotted with antibodies to myc, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. Note the increase in LC3II and p62 in VCP siRNA KD and mutant-expressing cells.(D) Fluorescent microscopy images of tetracycline-inducible U20S cells expressing GFP-LC3 treated with and without Baf for 4 h (DMSO control [left] or Baf+ [right]). Cells are control U20S cells and VCP-WT, -RH, -AE, and -EQ. (E) The number of GFP-LC3 puncta/cell was counted for control U20S and VCP-WT, -EQ, -RH, and -AE with and without Baf for 4 h (DMSO or Baf). Note the increase in basal GFP-LC3 puncta in mutant-expressing cells. (A-C and E) Error bars represent the standard error from four independent experiments. *, P < 0.01; and **, P < 0.001. Bars, 25 µm.


Figure 3 . ( A ) Epifluorescent images for GFP and mRFP in U20S or VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells transfected with mRFP - GFP - LC3 ( tfLC3 ) and treated with rapamycin for 2 h to induce autolysosome formation . ( B ) siRNA control ( Scr ) or VCP - KD - or Baf - treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation . ( A and B ) Open arrows denote autophagosomes ( both GFP and mRFP fluorescence ) , whereas closed arrows highlight autolysosomes ( mRFP only fluorescence ) . ( C ) The graph represents Pearson ' s coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments . Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 001 . ( D ) Lysates from U20S or tetracycline - inducible VCP - WT , - RH , or - AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α - tubulin . Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant ( RH and AE ) - expressing cells . Bars , 15 µm .

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Figure 3.(A) Epifluorescent images for GFP and mRFP in U20S or VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with mRFP-GFP-LC3 (tfLC3) and treated with rapamycin for 2 h to induce autolysosome formation. (B) siRNA control (Scr) or VCP-KD- or Baf-treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation. (A and B) Open arrows denote autophagosomes (both GFP and mRFP fluorescence), whereas closed arrows highlight autolysosomes (mRFP only fluorescence). (C) The graph represents Pearson's coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.(D) Lysates from U20S or tetracycline-inducible VCP-WT, -RH, or -AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α-tubulin. Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant (RH and AE)-expressing cells. Bars, 15 µm.


Figure 4 . ( A ) Lysates from siRNA - treated U20S cells ( scramble control [ Scr ] or VCP siRNA KD ) after autophagic induction via nutrient deprivation for 0 , 2 , or 4 h and immunoblotting for LC3 and α - tubulin . LC3II degrades over time in control but not in KD cells . One of two experiments is shown . ( B ) Lysates from U20S or tetracycline - inducible VCP - WT , - EQ , - RH , or - AE cells after autophagic induction via nutrient deprivation for 0 , 2 , 4 , or 6 h and immunoblotting for LC3 and α - tubulin . LC3II degrades in control and VCP - WT but degrades less efficiently in mutant ( EQ , RH , or AE ) - expressing cells . ( C ) Graphical representation of densitometric evaluation of LC3II and α - tubulin at each time point from three independent experiments .

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 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

Figure 4.(A) Lysates from siRNA-treated U20S cells (scramble control [Scr] or VCP siRNA KD) after autophagic induction via nutrient deprivation for 0, 2, or 4 h and immunoblotting for LC3 and α-tubulin. LC3II degrades over time in control but not in KD cells. One of two experiments is shown.(B) Lysates from U20S or tetracycline-inducible VCP-WT, -EQ, -RH, or -AE cells after autophagic induction via nutrient deprivation for 0, 2, 4, or 6 h and immunoblotting for LC3 and α-tubulin. LC3II degrades in control and VCP-WT but degrades less efficiently in mutant (EQ, RH, or AE)-expressing cells. (C) Graphical representation of densitometric evaluation of LC3II and α-tubulin at each time point from three independent experiments.


Figure 5 . ( A ) Epifluorescent images for GFP - LC3 ( LC3 ) and LTR of VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells transfected with GFP - LC3 and treated with rapamycin for 2 h to induce autolysosome formation . Open arrows highlight autolysosomes ( GFP and LTR colocalized ) , and closed arrows show autophagosomes ( GFP only ) . ( B ) Pearson ' s coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments . ( C ) Epifluorescent images for GFP - LC3 and endogenous Lamp1 immunohistochemistry of U20S , VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells or U20S cells cotreated with Baf transfected with GFP - LC3 and treated with rapamycin for 2 h to induce autolysosome formation . Arrows highlight autolysosomes ( GFP and Lamp1 colocalized ) . ( A and C ) The boxed regions in the merge field are enlarged in the adjacent panels . ( D ) Pearson ' s coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments . ( B and D ) Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 001 . ( E ) Electron microscopy of tetracycline - inducible control or VCP - WT - , VCP - RH - , or VCP - AE - expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h . Note the accumulation of autophagic structures in the mutant - expressing cell lines . Arrows denote autophagic structures .

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Figure 5.(A) Epifluorescent images for GFP-LC3 (LC3) and LTR of VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Open arrows highlight autolysosomes (GFP and LTR colocalized), and closed arrows show autophagosomes (GFP only). (B) Pearson's coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments.(C) Epifluorescent images for GFP-LC3 and endogenous Lamp1 immunohistochemistry of U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells or U20S cells cotreated with Baf transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Arrows highlight autolysosomes (GFP and Lamp1 colocalized). (A and C) The boxed regions in the merge field are enlarged in the adjacent panels. (D) Pearson's coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments. (B and D) Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.(E) Electron microscopy of tetracycline-inducible control or VCP-WT-, VCP-RH-, or VCP-AE-expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h. Note the accumulation of autophagic structures in the mutant-expressing cell lines. Arrows denote autophagic structures.


Figure 6 . ( A ) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or - 2 antibodies . Quadriceps section from a 9 - mo - oldVCP - WT mouse , age - matched VCP - RH transgenic , 12 - mo - oldVCP - RH transgenic , and IBMPFD patientmuscle biopsy are shown . Open arrows highlight p62 and Lamp1 / 2 colocalization , and closed arrows show regions of only p62immunofluorescence . Single myofibers containing RVs are outlined in the merge panels . ( B and C ) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines ( B shows RH12 , and C shows RH9 lines ) of 12 - mo - old VCP - RH - expressing transgenic mice . Note the large vacuolated structures that are perinuclear or subsarcolemmal . Bars : ( A ) 30 µm ; ( B and C ) 500 nm .

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Figure 6.(A) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or -2 antibodies. Quadriceps section from a 9-mo-oldVCP-WT mouse, age-matched VCP-RH transgenic, 12-mo-oldVCP-RH transgenic, and IBMPFD patientmuscle biopsy are shown. Open arrows highlight p62 and Lamp1/2 colocalization, and closed arrows show regions of only p62immunofluorescence. Single myofibers containing RVs are outlined in the merge panels.(B and C) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines (B shows RH12, and C shows RH9 lines) of 12-mo-old VCP-RH-expressing transgenic mice. Note the large vacuolated structures that are perinuclear or subsarcolemmal. Bars: (A) 30 µm; (B and C) 500 nm.


Figure 7 . ( A ) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH - expressing mice ( RH12 ) 2 d after electroporation ( baseline ) or after 24 h of nutrient deprivation ( starvation ) of polyQ80 ( Q80 ) - or polyQ19 - luciferase ( Q19 ) in the right and left tibialis anterior , respectively . The ratio of polyQ80 - luciferase / polyQ19 - luciferase is indicated below each image set . Values are in × 104 photons per second per square centimeter per steradian . ( B ) Box and whisker plot of the change ( Δ ) in the ratio of polyQ80 - luciferase / polyQ19 - luciferase activity in the left and right tibialis anterior muscle of control , VCP - WT , or one of two VCP - RH ( RH12 or RH9 ) transgenic mouse lines after 24 h of starvation . The graph is representative of three animals per group . The p - value for RH12 was 0 . 05 and 0 . 06 when compared with control animals or VCP - WT transgenic . The p - value for RH9 was 0 . 03 when compared with either control or VCP - WT transgenic . The p - value was 0 . 01 and 0 . 02 when combined RH12 and RH9 animals were compared with control or VCP - WT groups , respectively . There was no statistical difference between control or VCP - WT groups .

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Figure 7.(A) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH-expressing mice (RH12) 2 d after electroporation (baseline) or after 24 h of nutrient deprivation (starvation) of polyQ80 (Q80)- or polyQ19-luciferase (Q19) in the right and left tibialis anterior, respectively. The ratio of polyQ80-luciferase/polyQ19-luciferase is indicated below each image set. Values are in ×104 photons per second per square centimeter per steradian. (B) Box and whisker plot of the change (Δ) in the ratio of polyQ80-luciferase/polyQ19-luciferase activity in the left and right tibialis anterior muscle of control, VCP-WT, or one of two VCP-RH (RH12 or RH9) transgenic mouse lines after 24 h of starvation. The graph is representative of three animals per group. The p-value for RH12 was 0.05 and 0.06 when compared with control animals or VCP-WT transgenic. The p-value for RH9 was 0.03 when compared with either control or VCP-WT transgenic. The p-value was 0.01 and 0.02 when combined RH12 and RH9 animals were compared with control or VCP-WT groups, respectively. There was no statistical difference between control or VCP-WT groups.


Figure 8 . ( A ) Quantitation of mCherry - TDP - 43 distribution ( nuclear only or cytoplasmic ) in control U20S , VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells , or control U20S cells treated with 10 µM Baf or 50 µM chloroquine ( Chlq ) for 4 h from 10 different fields from two independent experiments . Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 02 when compared with VCP - WT - expressing cells . ( B ) Epifluorescent images for mCherry - TDP - 43 ( red ) and DAPI ( blue ) in control U20S , VCP - WT - , VCP - RH - , or VCP - AE - expressing cells , or control U20S cells treated with Baf or chloroquine for 4 h . Arrows denote cytosolic TDP - 43 and perinuclear TDP - 43 inclusions . ( C , top ) Immunoblot for TDP - 43 , lamin A / C , and actin from nuclear or cytosolic lysate fractions of mCherry - TDP - 43 - transfected VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells . Note the increase in cytosolic TDP - 43 from IBMPFD mutant - and EQ - expressing cells . ( bottom ) Immunoblot for TDP - 43 , lamin A / C , and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry - TDP - 43 or similarly transfected cells treated with 30 µg / ml ( cq1 ) or 120 µg / ml ( cq2 ) chloroquine diphosphate or 200 ng / ml Baf . Data are representative of three independent experiments . Bar , 15 µm .

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Figure 8.(A) Quantitation of mCherry-TDP-43 distribution (nuclear only or cytoplasmic) in control U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells, or control U20S cells treated with 10 µM Baf or 50 µM chloroquine (Chlq) for 4 h from 10 different fields from two independent experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.02 when compared with VCP-WT-expressing cells. (B) Epifluorescent images for mCherry-TDP-43 (red) and DAPI (blue) in control U20S, VCP-WT-, VCP-RH-, or VCP-AE-expressing cells, or control U20S cells treated with Baf or chloroquine for 4 h. Arrows denote cytosolic TDP-43 and perinuclear TDP-43 inclusions.(C, top) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of mCherry-TDP-43-transfected VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells. Note the increase in cytosolic TDP-43 from IBMPFD mutant- and EQ-expressing cells. (bottom) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry-TDP-43 or similarly transfected cells treated with 30 µg/ml (cq1) or 120 µg/ml (cq2) chloroquine diphosphate or 200 ng/ml Baf. Data are representative of three independent experiments. Bar, 15 µm.


Figure 9 . ( A ) TDP - 43 immunostaining of quadriceps skeletal muscle from 15 - mo - old control ( Cont ) or VCP - WT , - RH9 , or - RH12 transgenic mice . A single myofiber is outlined in white . ( B ) TDP - 43 immunostaining of quadriceps skeletal muscle from 3 - mo - old mice treated with saline or 50 mg / kg / d intraperitoneal chloroquine ( Chlq ) for 4 wk . ( A and B ) Insets show one myonuclei from each field . Bars , 30 µm .

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Figure 9.(A) TDP-43 immunostaining of quadriceps skeletal muscle from 15-mo-old control (Cont) or VCP-WT, -RH9, or -RH12 transgenic mice. A single myofiber is outlined in white.(B) TDP-43 immunostaining of quadriceps skeletal muscle from 3-mo-old mice treated with saline or 50 mg/kg/d intraperitoneal chloroquine (Chlq) for 4 wk. (A and B) Insets show one myonuclei from each field. Bars, 30 µm.