Figure 2Dissociation of SPIN90 - Arp2 / 3 - nucleated filaments . ( B ) The fraction of filaments still attached to the surface , versus time , for different concentrations of GST - NWASP - VCA . Black lines are exponential fits . Dissociation of SPIN90 - Arp2 / 3 - nucleated filaments . ( C ) Detachment rates , determined by exponential fits of survival curves ( as in B ) as a function of the concentration of VCA motifs from different NPFs . The data are fitted by a Michaelis - Menten equation ( resulting in an apparent KD of 0 . 10 ± 0 . 01 , 0 . 65 ± 0 . 36 and 0 . 82 ± 0 . 14 µM , for N - WASP , WASH and WASP , respectively ; and a maximum detachment rate of 0 . 033 ± 0 . 001 , 0 . 031 ± 0 . 006 , 0 . 010 ± 0 . 001 s - 1 for WASH , N - WASP and WASP , respectively ) . Dissociation of Arp2 / 3 - mediated branches . ( E ) The fraction of remaining branches , versus time , for different concentrations of GST - NWASP - VCA . Black lines are exponential fits . Dissociation of SPIN90 - Arp2 / 3 - nucleated filaments . ( A ) Schematic of the detachment experiment . Actin filaments were first nucleated from a SPIN90 - Arp2 / 3 - decorated surface , and elongated . Buffer with different concentrations of VCA was flowed in , and filament detachment events were monitored over time . Dissociation of Arp2 / 3 - mediated branches . ( F ) Debranching rates , determined by exponential fits of survival curves ( as in E ) as a function of the concentration of VCA motifs from different NPFs . The data are fitted by a Michaelis - Menten equation ( resulting in an apparent KD of 0 . 57 ± 0 . 35 , 0 . 81 ± 0 . 36 and 0 . 53 ± 0 . 23 µM , for WASH , N - WASP and WASP , respectively ; and a maximum detachment rate of 0 . 020 ± 0 . 004 , 0 . 012 ± 0 . 002 , 0 . 005 ± 0 . 001 s - 1 for WASH , N - WASP and WASP , respectively ) .

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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Dissociation of SPIN90-Arp2/3-nucleated filaments. (B) The fraction of filaments still attached to the surface, versus time, for different concentrations of GST-NWASP-VCA. Black lines are exponential fits.Dissociation of SPIN90-Arp2/3-nucleated filaments. (C) Detachment rates, determined by exponential fits of survival curves (as in B) as a function of the concentration of VCA motifs from different NPFs. The data are fitted by a Michaelis-Menten equation (resulting in an apparent KD of 0.10±0.01, 0.65±0.36 and 0.82±0.14 µM, for N-WASP, WASH and WASP, respectively; and a maximum detachment rate of 0.033± 0.001, 0.031± 0.006, 0.010± 0.001 s-1 for WASH, N-WASP and WASP, respectively).Dissociation of Arp2/3-mediated branches. (E) The fraction of remaining branches, versus time, for different concentrations of GST-NWASP-VCA. Black lines are exponential fits.Dissociation of SPIN90-Arp2/3-nucleated filaments. (A) Schematic of the detachment experiment. Actin filaments were first nucleated from a SPIN90-Arp2/3-decorated surface, and elongated. Buffer with different concentrations of VCA was flowed in, and filament detachment events were monitored over time. Dissociation of Arp2/3-mediated branches. (F) Debranching rates, determined by exponential fits of survival curves (as in E) as a function of the concentration of VCA motifs from different NPFs. The data are fitted by a Michaelis-Menten equation (resulting in an apparent KD of 0.57±0.35, 0.81±0.36 and 0.53±0.23 µM, for WASH, N-WASP and WASP, respectively; and a maximum detachment rate of 0.020± 0.004, 0.012± 0.002, 0.005± 0.001 s-1 for WASH, N-WASP and WASP, respectively).


Figure 3A . The fraction of filaments still attached to the surface by SPIN90 - Arp2 / 3 , over time , when exposed to different concentrations of GMF . For each experiment , n = 36 - 43 filaments . Black lines are exponential fits . Each experiment was repeated 3 times ( technical replicates ) , yielding similar results . B . Detachment rates , determined by exponential fits of survival curves shown in A , as a function of the concentration of GMF . The error bars are determined by fitting the edges of the 95 % confidence intervals ( not shown , for readability ) of the survival curves . The data are fitted by a Michaelis - Menten equation , yielding an apparent dissociation constant KD = 70 ± 23 nM and a maximum detachment rate of 0 . 017 ± 0 . 001s - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. The fraction of filaments still attached to the surface by SPIN90-Arp2/3, over time, when exposed to different concentrations of GMF. For each experiment, n=36-43 filaments. Black lines are exponential fits. Each experiment was repeated 3 times (technical replicates), yielding similar results.B. Detachment rates, determined by exponential fits of survival curves shown in A, as a function of the concentration of GMF. The error bars are determined by fitting the edges of the 95% confidence intervals (not shown, for readability) of the survival curves. The data are fitted by a Michaelis-Menten equation, yielding an apparent dissociation constant KD=70±23 nM and a maximum detachment rate of 0.017±0.001s-1.


Figure 4Assessing the presence of Arp2 / 3 on SPIN90 after filament departure . ( B - C ) The fraction of SPIN90 that re - nucleated a filament 500 seconds after the detachment of the initial filament , was quantified for different conditions . GMF and VCA interfere with the SPIN90 - Arp2 / 3 interaction , in the absence of filament . ( G ) Immuno - blots from pull - down assays where GST beads , decorated with GST or GST - SPIN90 - Cter ( the functional domain of SPIN90 ) , were incubated with 0 . 4 μΜ Arp2 / 3 and gradient concentrations of GMF or VCA for 1 hour at room temperature . After the unbound protein was washed out , the amount of Arp2 / 3 attached to the beads was detected by Anti - ArpC2 antibody . Ponceau red straining of the membrane verified that the beads were loaded with equal amounts of GST or GST - SPIN90 ( Appendix Figure S2 ) . GMF and VCA interfere with the SPIN90 - Arp2 / 3 interaction , in the absence of filament . ( H - I ) Quantification of the amount of ArpC2 remaining on the beads , in different conditions .

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 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 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

Figure 4Assessing the presence of Arp2/3 on SPIN90 after filament departure. (B-C) The fraction of SPIN90 that re-nucleated a filament 500 seconds after the detachment of the initial filament, was quantified for different conditions.GMF and VCA interfere with the SPIN90-Arp2/3 interaction, in the absence of filament. (G) Immuno-blots from pull-down assays where GST beads, decorated with GST or GST-SPIN90-Cter (the functional domain of SPIN90), were incubated with 0.4 μΜ Arp2/3 and gradient concentrations of GMF or VCA for 1 hour at room temperature. After the unbound protein was washed out, the amount of Arp2/3 attached to the beads was detected by Anti-ArpC2 antibody. Ponceau red straining of the membrane verified that the beads were loaded with equal amounts of GST or GST-SPIN90 (Appendix Figure S2).GMF and VCA interfere with the SPIN90-Arp2/3 interaction, in the absence of filament. (H-I) Quantification of the amount of ArpC2 remaining on the beads, in different conditions.


Figure 5A - C . Survival fractions for branches exposed to different forces in the microfluidics chamber , while being exposed to 0 . 15µM G - actin , alone ( gray curves , A ) , with 1 µM VCA ( blue curves , B ) , or with 1 µM GMF ( red curves , C ) . The shaded areas represent 95 % confidence intervals . Black lines are exponential fits . D . Summary of the debranching rates , resulting from the exponential fits in ( A - C ) . E - G . Survival fractions for SPIN90 - Arp2 / 3 - nucleated filaments exposed to different forces in the microfluidics chamber , while being exposed to 0 . 15µM G - actin , alone ( gray curves , E ) , with 0 . 1 µM VCA ( blue curves , F ) , or with 0 . 1 µM GMF ( red curves , G ) . The shaded areas represent 95 % confidence intervals . Black lines are exponential fits . H . Summary of the detachment rates for SPIN90 - Arp2 / 3 - nucleated filaments , resulting from the exponential fits in ( E - G ) .

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

Figure 5A-C. Survival fractions for branches exposed to different forces in the microfluidics chamber, while being exposed to 0.15µM G-actin, alone (gray curves, A), with 1 µM VCA (blue curves, B), or with 1 µM GMF (red curves, C). The shaded areas represent 95% confidence intervals. Black lines are exponential fits.D. Summary of the debranching rates, resulting from the exponential fits in (A-C).E-G. Survival fractions for SPIN90-Arp2/3-nucleated filaments exposed to different forces in the microfluidics chamber, while being exposed to 0.15µM G-actin, alone (gray curves, E), with 0.1 µM VCA (blue curves, F), or with 0.1 µM GMF (red curves, G). The shaded areas represent 95% confidence intervals. Black lines are exponential fits.H. Summary of the detachment rates for SPIN90-Arp2/3-nucleated filaments, resulting from the exponential fits in (E-G).


Figure 6A . Fraction of remaining branches , over time , starting at an average of 5 and 25 minutes after branch nucleation , in the presence ( blue ) or absence ( gray ) of VCA . Lines are exponential fits , yielding debranching rates of ( 3 . 7 ± 0 . 6 ) x10 - 4 s - 1 and ( 20 ± 8 ) x10 - 4 s - 1 without VCA for 5 - and 25 - minute - old branches , respectively ; and debranching rates of ( 3 . 8 ± 1 . 3 ) x10 - 3 s - 1 and ( 8 . 2 ± 2 . 8 ) x10 - 3 s - 1 with 0 . 5 µM VCA for 5 - and 25 - minute - old branches , respectively . B . Fraction of attached SPIN90 - nucleated filaments , over time , starting at an average of 5 and 25 minutes after branch nucleation , in the presence ( blue ) or absence ( gray ) of VCA . Lines are exponential fits , yielding detachment rates of ( 12 ± 7 ) x10 - 4 s - 1 and ( 7 ± 3 ) x10 - 4 s - 1 without VCA for 5 - and 25 - minute - old filaments , respectively ( p = 0 . 2 , logrank test ) ; and detachment rates of ( 2 . 5 ± 1 . 1 ) x10 - 2 s - 1 and ( 3 . 1 ± 1 . 7 ) x10 - 2 s - 1 with 1 µM VCA for 5 - and 25 - minute - old filaments , respectively ( p = 0 . 4 , logrank test ) .

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

Figure 6A. Fraction of remaining branches, over time, starting at an average of 5 and 25 minutes after branch nucleation, in the presence (blue) or absence (gray) of VCA. Lines are exponential fits, yielding debranching rates of (3.7±0.6)x10-4 s-1 and (20±8)x10-4 s-1 without VCA for 5- and 25-minute-old branches, respectively; and debranching rates of (3.8±1.3)x10-3 s-1 and (8.2±2.8)x10-3 s-1 with 0.5 µM VCA for 5- and 25-minute-old branches, respectively.B. Fraction of attached SPIN90-nucleated filaments, over time, starting at an average of 5 and 25 minutes after branch nucleation, in the presence (blue) or absence (gray) of VCA. Lines are exponential fits, yielding detachment rates of (12±7)x10-4 s-1 and (7±3)x10-4 s-1 without VCA for 5- and 25-minute-old filaments, respectively (p=0.2, logrank test); and detachment rates of (2.5±1.1)x10-2 s-1 and (3.1±1.7)x10-2 s-1 with 1 µM VCA for 5- and 25-minute-old filaments, respectively (p=0.4, logrank test).


Figure 7A . The fraction of remaining branches , versus time , exposed to 0 . 16 µM G - actin ( black ) supplemented with 50 nM cortactin ( red ) . The branch junctions were exposed to an average force of 1 . 4 pN . B . The fraction of remaining SPIN90 - Arp2 / 3 - nucleated filaments , versus time , exposed to 0 . 15 µM G - actin ( black ) supplemented with 10 nM cortactin ( red ) . C . The fraction of remaining SPIN90 - Arp2 / 3 - nucleated filaments , versus time , exposed to 0 . 15 µM G - actin , with 0 . 5 µM VCA ( black ) supplemented with 10 nM cortactin ( red ) .

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 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

Figure 7A. The fraction of remaining branches, versus time, exposed to 0.16 µM G-actin (black) supplemented with 50 nM cortactin (red). The branch junctions were exposed to an average force of 1.4 pN.B. The fraction of remaining SPIN90-Arp2/3-nucleated filaments, versus time, exposed to 0.15 µM G-actin (black) supplemented with 10 nM cortactin (red).C. The fraction of remaining SPIN90-Arp2/3-nucleated filaments, versus time, exposed to 0.15 µM G-actin, with 0.5 µM VCA (black) supplemented with 10 nM cortactin (red).


figf1 ( a ) p62 localizes to the MR during cytokinesis and abscission , and is taken up together with the MR by one daughter cell . U2OS cells were fixed and stained with anti - p62 ( red ) and anti - MKLP1 ( green ) antibodies . Scale bars are 10 μm . Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division . The arrowheads point to MRs , which are shown enlarged in the insets . The quantification is presented in Supplementary Information Fig . S1a . ( b ) siRNA - mediated depletion of p62 from U2OS cells results in moderate MR accumulation , compared with a control depletion ( lamin A / C ) . For quantification , MRs were counted in control cells and in p62 - depleted cells that were negative for p62 staining . Data are means ± s . d . of three independent experiments ( n , total number of cells counted ; P , probability of a two - tailed paired t - test ) .

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 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

figf1(a) p62 localizes to the MR during cytokinesis and abscission, and is taken up together with the MR by one daughter cell. U2OS cells were fixed and stained with anti-p62 (red) and anti-MKLP1 (green) antibodies. Scale bars are 10 μm. Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division. The arrowheads point to MRs, which are shown enlarged in the insets. The quantification is presented in Supplementary Information Fig. S1a.(b) siRNA-mediated depletion of p62 from U2OS cells results in moderate MR accumulation, compared with a control depletion (lamin A/C). For quantification, MRs were counted in control cells and in p62-depleted cells that were negative for p62 staining. Data are means ± s.d. of three independent experiments (n, total number of cells counted; P, probability of a two-tailed paired t-test).


figf2 ( a ) Atg8 ( LC3 ) localizes to the MR during abscission . The upper panels show stills from time - lapse microscopy of GFP - Atg8 - expressing cells . The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission . The enlarged clipping on the right shows the midbody area at time - point 68 min . Scale bar is 10 μm . ( b ) Cytosolic MRs are found inside Atg8 - positive vesicles . U2OS cells stably expressing GFP - Atg8 were fixed and stained for MKLP1 ( red ) and α - tubulin ( blue ) . Scale bars are 10 μm and 1 μm for the enlarged boxed areas . Putative different stages of autophagosomes ( left ) and lysosomes ( right ) are shown . ( c ) Juxtanuclear MRs are found in Rab7 - and LAMP2 - positive lysosomes . U2OS cells stably expressing DsRed - Rab7 were fixed and stained for MKLP1 α - tubulin and DAPI ( left panels ) ; wild - type U2OS were stained for MKLP1 and LAMP2 ( right panel ) . Scale bars are 10 μm . The insets are enlargements of the engulfed MRs . Scale bars are 1 μm .

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

figf2(a) Atg8 (LC3) localizes to the MR during abscission. The upper panels show stills from time-lapse microscopy of GFP-Atg8-expressing cells. The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission. The enlarged clipping on the right shows the midbody area at time-point 68 min. Scale bar is 10 μm.(b) Cytosolic MRs are found inside Atg8-positive vesicles. U2OS cells stably expressing GFP-Atg8 were fixed and stained for MKLP1 (red) and α-tubulin (blue). Scale bars are 10 μm and 1 μm for the enlarged boxed areas. Putative different stages of autophagosomes (left) and lysosomes (right) are shown.(c) Juxtanuclear MRs are found in Rab7- and LAMP2-positive lysosomes. U2OS cells stably expressing DsRed-Rab7 were fixed and stained for MKLP1 α-tubulin and DAPI (left panels); wild-type U2OS were stained for MKLP1 and LAMP2 (right panel). Scale bars are 10 μm. The insets are enlargements of the engulfed MRs. Scale bars are 1 μm.


figf3 ( a ) Accumulation of MRs in cells treated with the PI ( 3 ) K inhibitor 3 - MA . Left panels show HeLa cells treated with 3 - MA ( 4 mM ) for 3 days . Cells were fixed and stained . Images are colour - inverted for better visualization . The quantification of this experiment is shown on the upper right ( mean ± s . d . of three independent experiments ; n = total number of cells counted ) . The lower right panel shows an enlarged area of 3 - MA - treated cells in true colour . Scale bars are 20 μm . ( b ) siRNA - mediated depletion of Atg6 ( Beclin - 1 ) and Atg7 from HeLa cells causes MR accumulations . The right panel shows the quantification of MRs per cell ( mean ± s . d . of three independent experiments ) ; the left panel the efficiency of Atg6 and Atg7 depletion . ( c ) Inhibition of lysosomal acidification leads to MR accumulation . Panels on the left show HeLa cells left untreated or treated with chloroquine ( 20 μM ) or ammonium chloride ( 10 mM ) for 3 days . Scale bar are 20 μm . Cells were fixed and stained as indicated . A quantification of this experiment is shown on the left ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

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 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

figf3(a) Accumulation of MRs in cells treated with the PI(3)K inhibitor 3-MA. Left panels show HeLa cells treated with 3-MA (4 mM) for 3 days. Cells were fixed and stained. Images are colour-inverted for better visualization. The quantification of this experiment is shown on the upper right (mean ± s.d. of three independent experiments; n = total number of cells counted). The lower right panel shows an enlarged area of 3-MA-treated cells in true colour. Scale bars are 20 μm.(b) siRNA-mediated depletion of Atg6 (Beclin-1) and Atg7 from HeLa cells causes MR accumulations. The right panel shows the quantification of MRs per cell (mean ± s.d. of three independent experiments); the left panel the efficiency of Atg6 and Atg7 depletion.(c) Inhibition of lysosomal acidification leads to MR accumulation. Panels on the left show HeLa cells left untreated or treated with chloroquine (20 μM) or ammonium chloride (10 mM) for 3 days. Scale bar are 20 μm. Cells were fixed and stained as indicated. A quantification of this experiment is shown on the left (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


figf4 ( a ) Cell - cycle lengths of different cell lines correlate well with the average amount of MRs . Indicated cell lines were grown on coverslips , fixed and MRs were counted after staining with anti - MKLP1 antibodies . Cell - cycle lengths were assessed as described in the Methods . Data are mean ± s . d . of three independent experiments . ( b ) The degree of MR persistence depends on the time and not on cell - cycle duration . HeLa cells were either left untreated or were arrested in S - phase with hydroxyurea ( HU , 5 mM ) . Cells were fixed and stained as indicated . Scale bars are 20 μm . The right panel shows quantification of MR counting ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

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

figf4(a) Cell-cycle lengths of different cell lines correlate well with the average amount of MRs. Indicated cell lines were grown on coverslips, fixed and MRs were counted after staining with anti-MKLP1 antibodies. Cell-cycle lengths were assessed as described in the Methods. Data are mean ± s.d. of three independent experiments.(b) The degree of MR persistence depends on the time and not on cell-cycle duration. HeLa cells were either left untreated or were arrested in S-phase with hydroxyurea (HU, 5 mM). Cells were fixed and stained as indicated. Scale bars are 20 μm. The right panel shows quantification of MR counting (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


figf5 ( a ) Fibroblasts of individuals with mucolipidosis II ( I - cell disease ; upper right panel ) or Hurler syndrome ( lower right panel ) were compared with parental control cells ( left panels ) . The alleles are indicated . Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA . Arrowheads point to the accumulated MRs . Scale bars are 20 μm . ( b ) Quantification of a ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

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

figf5(a) Fibroblasts of individuals with mucolipidosis II (I-cell disease; upper right panel) or Hurler syndrome (lower right panel) were compared with parental control cells (left panels). The alleles are indicated. Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA. Arrowheads point to the accumulated MRs. Scale bars are 20 μm. (b) Quantification of a (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


Figure 1A Expression patterns of pSEU : : SEU - GFP and pWOX5 : : GFP in the embryos of the indicated genotypes at dermatogen , early globular , and heart stages . White arrows indicate the QC precursor cell , and white dashed lines indicate embryos . Scale bars : 10 µm . B Quantification of pWOX5 : : GFP GFP fluorescence in wild - type ( WT ) and seu - 3 mutant embryos . Fluorescence intensity at the early globular stage of WT embryos was set to 1 . Data Information : In ( B ) data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . C , D Expression pattern of pWOX5 : : GFP in WT ( C ) and seu - 3 ( D ) embryos at 5 days after germination ( DAG ) . Scale bars : 20 µm . E Quantification of GFP fluorescence in the QC of pWOX5 : : GFP transgenic plants , as shown in ( C ) and ( D ) . Fluorescence intensity was normalized to the WT . Data Information : ( E ) , data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . F RT - qPCR analysis of the relative expression levels of WOX5 in WT and seu - 3 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data Information : ( F ) data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . G - J Modified pseudo - Schiff propidium iodide ( mPS - PI ) staining of stem cell niche areas in the indicated genotypes at 5 DAG . Blue arrows indicate the QC , and red arrows indicate the columella stem cells ( CSCs ) . The numbers denote total number of scored samples , with similar phenotypes showing in ( G - J ) . Scale bars : 20 μm . K Quantification of the CSC layer in the indicated genotypes at 5 DAG . Data Information ( K ) , data represent mean ± SD of three independent replicates . n denotes the total number of scored samples . Individual values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . L , M Double staining of the QC184 β - glucuronidase ( GUS ) marker ( light blue ) and starch granules ( dark brown ) in WT ( L ) and seu - 3 ( M ) seedlings at 5 DAG . In white arrows indicate the CSCs . Scale bars : 20 μm . N , O Expression pattern of J2341 in WT ( N ) and seu - 3 ( O ) seedlings at 5 DAG . P , Q Expression pattern of CS9227 in WT ( P ) and seu - 3 ( Q ) seedlings at 5 DAG . In white arrows indicate the CSCs . Scale bars : 20 μm .

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

Figure 1A Expression patterns of pSEU::SEU-GFP and pWOX5::GFP in the embryos of the indicated genotypes at dermatogen, early globular, and heart stages. White arrows indicate the QC precursor cell, and white dashed lines indicate embryos. Scale bars: 10 µm.B Quantification of pWOX5::GFP GFP fluorescence in wild-type (WT) and seu-3 mutant embryos. Fluorescence intensity at the early globular stage of WT embryos was set to 1. Data Information: In (B) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).C, D Expression pattern of pWOX5::GFP in WT (C) and seu-3 (D) embryos at 5 days after germination (DAG). Scale bars: 20 µm. E Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic plants, as shown in (C) and (D). Fluorescence intensity was normalized to the WT. Data Information: (E), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).F RT-qPCR analysis of the relative expression levels of WOX5 in WT and seu-3 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data Information: (F) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).G-J Modified pseudo-Schiff propidium iodide (mPS-PI) staining of stem cell niche areas in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indicate the columella stem cells (CSCs). The numbers denote total number of scored samples, with similar phenotypes showing in (G-J). Scale bars: 20 μm. K Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data Information (K), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).L, M Double staining of the QC184 β-glucuronidase (GUS) marker (light blue) and starch granules (dark brown) in WT (L) and seu-3 (M) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.N, O Expression pattern of J2341 in WT (N) and seu-3 (O) seedlings at 5 DAG. P, Q Expression pattern of CS9227 in WT (P) and seu-3 (Q) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.


Figure 2A - C Expression pattern of pWOX5 : : GFP in WT ( A ) , scr - 3 ( B ) and plt1 - 4 plt2 - 2 ( C ) at 5 DAG . D Quantification of GFP fluorescence in the QC of pWOX5 : : GFP transgenic seedlings , as shown in ( A - C ) . GFP signal intensity in each genotype was normalized relative to that in the WT . Data information : In ( D ) n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . In ( D ) different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) . E RT - qPCR analysis of the relative expression levels of WOX5 in WT , scr - 3 , and plt1 - 4 plt2 - 2 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data information : In ( E ) , * * P < 0 . 01 ( Student ' s t - test ) . Scale bars : 20 µm . F Primary root length of the indicated genotypes at 5 DAG . Data information : In ( F ) n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . In ( F ) , different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) . G - J Representative confocal images of the indicated genotypes at 5 DAG . White arrows indicate the QC . K Quantification of GFP fluorescence in the QC of pSCR : : GFP and pSCR : : GFP - SCR transgenic seedlings , as shown GFP signal intensity of each genotype was normalized relative to that of the WT . Data information ( K ) , n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . ( K ) , * * P < 0 . 01 ( Student ' s t - test ) . Scale bars : 20 µm . L - Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG . White dashed lines indicate the QC region . The numbers denote total number of scored samples , with similar phenotypes showing in ( L - Q ) .

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

Figure 2A-C Expression pattern of pWOX5::GFP in WT (A), scr-3 (B) and plt1-4 plt2-2 (C) at 5 DAG. D Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic seedlings, as shown in (A-C). GFP signal intensity in each genotype was normalized relative to that in the WT. Data information: In (D) n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (D) different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01).E RT-qPCR analysis of the relative expression levels of WOX5 in WT, scr-3, and plt1-4 plt2-2 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data information: In (E) , **P < 0.01 (Student's t-test). Scale bars: 20 µm.F Primary root length of the indicated genotypes at 5 DAG. Data information: In (F) n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (F), different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01).G-J Representative confocal images of the indicated genotypes at 5 DAG. White arrows indicate the QC.K Quantification of GFP fluorescence in the QC of pSCR::GFP and pSCR::GFP-SCR transgenic seedlings, as shown GFP signal intensity of each genotype was normalized relative to that of the WT. Data information (K), n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. (K), **P < 0.01 (Student's t-test). Scale bars: 20 µm.L-Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG. White dashed lines indicate the QC region. The numbers denote total number of scored samples, with similar phenotypes showing in (L-Q).


Figure 3A Yeast two - hybrid ( Y2H ) assays showing the interaction between SEU and SCR . The yeast transformants were plated on synthetic defined ( SD ) media lacking Leu and Trp ( SD / - 2 ) or lacking Ade , His , Leu , and Trp ( SD / - 4 ) to assess protein - protein interactions . AD , GAL4 activation domain ; BD , GAL4 DNA - binding domain . B In vitro pull - down assays showing that SEU directly interacts with SCR but not with SHR . SCR - FLAG was pulled down by SEU - MBP immobilized on amylose resin . Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti - FLAG antibody . The asterisk indicates the position of SEU - MBP . C Verification of in vivo interactions between SCR and SEU in N . benthamiana leaves via Co - IP assays . SCR - GFP and SEU - myc were transiently coexpressed in N . benthamiana leaves . Protein samples were immunoprecipitated using anti - myc antibody . D Co - IP assays of SEU with SCR in Arabidopsis . Protein extracts from WT and SCR - GFP roots were isolated at 5 DAG and immunoprecipitated with anti - GFP antibody . F , G SCR and SEU physically bind to the WOX5 promoter , as shown by ChIP - qPCR analysis . Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP antibody . The precipitated DNA was used as a template for qPCR analysis . A , B , and I indicated the PCR amplicons ACT7 , control . Data information : In ( F ) , ( G ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) . H Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter , as shown by ChIP - qPCR analysis . Chromatin was extracted from SEU - GFP and SEU - GFP scr - 3 seedlings at 5 DAG and precipitated with anti - GFP antibody . ChIP signals were quantified by qPCR as a percentage of total input DNA . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) . I SEU stimulated SCR - mediated WOX5 promoter activation in transient expression assays in N . benthamiana leaves . The pWOX5 : : LUC reporter was cotransformed with the indicated effector constructs . The pWOX5 : : LUC activity was normalized relative to the internal control ( LUC / Renilla luciferase [ REN ] ) . The schematic diagram shows the construct used in the transient expression assays . Arrows indicate promoter regions , and boxes indicate coding sequences . Different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) . J - M Representative confocal images of the indicated genotypes at 5 DAG . Scale bars : 20 μm . Insets show the QC region in which the solid white lines indicate the QC of WT , and the dashed white lines indicate the QC of seu - 3 . ( Insets scale bars : 5 μm ) . N Quantification of GFP signal intensity in the QC of WT and seu - 3 seedlings expressing pWOX5 : : SEU - GFP and pWOX5 : : SCR - GFP . GFP signal intensity in seu - 3 seedlings was normalized relative to the WT . Individual values ( black dots ) are shown . n denotes the total number of scored samples . Data information : In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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

Figure 3A Yeast two-hybrid (Y2H) assays showing the interaction between SEU and SCR. The yeast transformants were plated on synthetic defined (SD) media lacking Leu and Trp (SD/-2) or lacking Ade, His, Leu, and Trp (SD/-4) to assess protein-protein interactions. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain.B In vitro pull-down assays showing that SEU directly interacts with SCR but not with SHR. SCR-FLAG was pulled down by SEU-MBP immobilized on amylose resin. Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti-FLAG antibody. The asterisk indicates the position of SEU-MBP.C Verification of in vivo interactions between SCR and SEU in N. benthamiana leaves via Co-IP assays. SCR-GFP and SEU-myc were transiently coexpressed in N. benthamiana leaves. Protein samples were immunoprecipitated using anti-myc antibody.D Co-IP assays of SEU with SCR in Arabidopsis. Protein extracts from WT and SCR-GFP roots were isolated at 5 DAG and immunoprecipitated with anti-GFP antibody.F, G SCR and SEU physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP antibody. The precipitated DNA was used as a template for qPCR analysis. A, B, and I indicated the PCR amplicons ACT7, control. Data information: In (F), (G) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).H Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was extracted from SEU-GFP and SEU-GFP scr-3 seedlings at 5 DAG and precipitated with anti-GFP antibody. ChIP signals were quantified by qPCR as a percentage of total input DNA. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).I SEU stimulated SCR-mediated WOX5 promoter activation in transient expression assays in N. benthamiana leaves. The pWOX5::LUC reporter was cotransformed with the indicated effector constructs. The pWOX5::LUC activity was normalized relative to the internal control (LUC/Renilla luciferase [REN]). The schematic diagram shows the construct used in the transient expression assays. Arrows indicate promoter regions, and boxes indicate coding sequences. Different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01). Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).J-M Representative confocal images of the indicated genotypes at 5 DAG. Scale bars: 20 μm. Insets show the QC region in which the solid white lines indicate the QC of WT, and the dashed white lines indicate the QC of seu-3. (Insets scale bars: 5 μm). N Quantification of GFP signal intensity in the QC of WT and seu-3 seedlings expressing pWOX5::SEU-GFP and pWOX5::SCR-GFP. GFP signal intensity in seu-3 seedlings was normalized relative to the WT. Individual values (black dots) are shown. n denotes the total number of scored samples. Data information: In (N), data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


Figure 4A Y2H assays showing that SEU interacts with SDG4 and SDG25 . The yeast transformants were plated on SD / - 2 and SD / - 4 media to assess protein - protein interactions . C SDG4 physically bind to the WOX5 promoter , as shown by ChIP - qPCR analysis . ACT7 , control . Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . The precipitated DNA was used as a template for qPCR analysis . In ( C ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . D , E ChIP - qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter . ChIP signal was normalized to the WT . Individual values ( black dots ) are shown . Data information : Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . The precipitated DNA was used as a template for qPCR analysis . In ( D ) , ( E ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . F , G Expression pattern of pWOX5 : : GFP in the indicated genotypes at 5 DAG . Scale bars : 20 µm . H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5 : : GFP . GFP signal intensity in the WT was set to 1 . n denotes the total number of scored samples . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . I RT - qPCR analysis of the relative expression levels of WOX5 in WT and ashr3 - 1 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Individual values ( black dots ) are shown . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . J - M mPS - PI staining of stem cell niche in the indicated genotypes at 5 DAG . Blue arrows indicate the QC , and red arrows indiate the CSCs . The numbers denote total number of scored samples , with same phenotypes showing in ( J - M ) . Scale bars : 10 μm . N Quantification of the CSC layer in the indicated genotypes at 5 DAG . Data information In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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 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 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 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 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

Figure 4A Y2H assays showing that SEU interacts with SDG4 and SDG25. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.C SDG4 physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. ACT7, control. Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (C) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).D, E ChIP-qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter. ChIP signal was normalized to the WT. Individual values (black dots) are shown. Data information: Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (D), (E) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).F, G Expression pattern of pWOX5::GFP in the indicated genotypes at 5 DAG. Scale bars: 20 µm. H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5::GFP. GFP signal intensity in the WT was set to 1. n denotes the total number of scored samples. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).I RT-qPCR analysis of the relative expression levels of WOX5 in WT and ashr3-1 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Individual values (black dots) are shown. Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).J-M mPS-PI staining of stem cell niche in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indiate the CSCs. The numbers denote total number of scored samples, with same phenotypes showing in (J-M). Scale bars: 10 μm. N Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data information In (N), data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


Figure 5A - G Representative confocal images of the indicated genotypes at 5 DAG . The white dashed lines indicate the QC region . Scale bars : 20 μm . H Domain mapping of SEU involved in interactions with SCR and SDG4 . The yeast transformants were plated on SD / - 2 and SD / - 4 media to assess protein - protein interactions . I SEU associates with SCR and SDG4 in N . benthamiana leaves , as shown by Co - IP assays . SDG4 - myc , SCR - HA , and SEU - GFP were coinfiltrated into N . benthamiana leaves . Protein samples were immunoprecipitated with anti - GFP antibody and immunoblotted with anti - myc and anti - HA antibody . N . benthamiana leaves coinfiltrated with SDG4 - myc and SCR - HA were used as a negative control . J Co - IP assays of SCR with SEU and SDG4 in Arabidopsis . Proteins extracted from SDG4 - myc and SDG4 - myc SCR - GFP plants were immunoprecipitated using anti - GFP antibody , and immunoblotted using anti - myc and anti - SEU antibodies .

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Figure 5A-G Representative confocal images of the indicated genotypes at 5 DAG. The white dashed lines indicate the QC region. Scale bars: 20 μm.H Domain mapping of SEU involved in interactions with SCR and SDG4. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.I SEU associates with SCR and SDG4 in N. benthamiana leaves, as shown by Co-IP assays. SDG4-myc, SCR-HA, and SEU-GFP were coinfiltrated into N. benthamiana leaves. Protein samples were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-myc and anti-HA antibody. N. benthamiana leaves coinfiltrated with SDG4-myc and SCR-HA were used as a negative control.J Co-IP assays of SCR with SEU and SDG4 in Arabidopsis. Proteins extracted from SDG4-myc and SDG4-myc SCR-GFP plants were immunoprecipitated using anti-GFP antibody, and immunoblotted using anti-myc and anti-SEU antibodies.


Figure 1 ( B ) Dot plot indicating fraction of tf - Reporters delivered to the lysosome by ATG7 - independent mechanisms . The median red : green ratio of each population was used to calculate fractional delivery according to the following formula : ( ratiosgATG7 - ratiosgATG9A ) / ( ratioNegativeCtl1 - ratiosgATG9A ) , with the assumption that sgATG9A yields a true autophagy - null phenotype . Mean values for each reporter are indicated by a black bar . n = 3 for each reporter . ( C ) Wild - type ( WT ) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 ( BafA1 ) or DMSO for 18h and analyzed by flow cytometry for red : green ratio of tf - NBR1 . Median values for each sample are identified by a black line within each violin . The red dotted line across all samples corresponds to the red : green ratio in wild - type cells . The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions ( ATG9AKO cells ) . n = 10 , 000 cells . ( D ) Representative confocal micrographs ( as maximum intensity projections ) of wild - type K562 cells and indicated deletion isolates expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence microscopy . Red fluorescent protein ( RFP ) , magenta ; Green fluorescent protein ( GFP ) , green ; Hoechst , blue . Scale bars : large panels , 5 µm ; small panels , 1 µm . All images scaled equally . ( E ) Correlative light and electron microscopy ( CLEM ) of K562 ATG7KO cells expressing tf - NBR1 under basal conditions . Analysis workflow is indicated by green arrows . White arrows indicate representative structures of interest . White boxes demarcate zoomed areas in subsequent images . NBR1 , green ; Hoechst , blue . Scale bar ( small images ) , 2 . 5 µm . Scale bar ( large images ) , 250 nm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 1(B) Dot plot indicating fraction of tf-Reporters delivered to the lysosome by ATG7-independent mechanisms. The median red:green ratio of each population was used to calculate fractional delivery according to the following formula: (ratiosgATG7-ratiosgATG9A)/(ratioNegativeCtl1-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Mean values for each reporter are indicated by a black bar. n=3 for each reporter.(C) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells.(D) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally.(E) Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm.


Figure 2 ( A ) Box plot of average beta scores ( similar to log - fold change ) for tf - NBR1 flux in indicated deletion cell lines . Median values ( central band ) , inner quartiles ( boxed regions ) , 1 . 5 interquartile ranges ( whiskers ) , and outliers ( dots ) are indicated . Representative hits across all three genetic backgrounds are indicated in blue . ( B ) Gene correlation plot of average beta scores for lipidation dependent autophagy ( tf - NDP52 , from ( Shoemaker et al , 2019 ) ) and lipidation independent autophagy ( average of tf - NBR1 from ATG7KO , ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0 . 5 across all three lipidation - deficient cell lines . In black are lipidation components . Dashed lines , top 1 % of beta scores . ( C ) Wild - type K562 cells and indicated deletion isolates expressing Cas9 and tf - NDP52 or tf - NBR1 were transduced with individual sgRNAs for indicated genes . The median red : green ratio of each population was used to calculate the fold - repression according to the following formula : ( ratiosgGene - ratiosgATG9A ) / ( ratioNegativeCtl - ratiosgATG9A ) , with the assumption that sgATG9A yields a true autophagy - null phenotype . Deeper shades of red indicate stronger suppressor phenotypes . Genes were clustered on the basis of their patterns of genetic interactions .

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 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 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

Figure 2(A) Box plot of average beta scores (similar to log-fold change) for tf-NBR1 flux in indicated deletion cell lines. Median values (central band), inner quartiles (boxed regions), 1.5 interquartile ranges (whiskers), and outliers (dots) are indicated. Representative hits across all three genetic backgrounds are indicated in blue.(B) Gene correlation plot of average beta scores for lipidation dependent autophagy (tf-NDP52, from (Shoemaker et al, 2019)) and lipidation independent autophagy (average of tf-NBR1 from ATG7KO, ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0.5 across all three lipidation-deficient cell lines. In black are lipidation components. Dashed lines, top 1% of beta scores.(C) Wild-type K562 cells and indicated deletion isolates expressing Cas9 and tf-NDP52 or tf-NBR1 were transduced with individual sgRNAs for indicated genes. The median red:green ratio of each population was used to calculate the fold-repression according to the following formula: (ratiosgGene-ratiosgATG9A)/(ratioNegativeCtl-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Deeper shades of red indicate stronger suppressor phenotypes. Genes were clustered on the basis of their patterns of genetic interactions.


Figure 3 ( A ) Wild - type ( WT ) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 ( BafA1 ) or DMSO for 18h and analyzed by flow cytometry for red : green ratio of tf - NBR1 . Median values for each sample are identified by a black line within each violin . The red dotted line across all samples corresponds to the red : green ratio in wild - type cells . The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions ( ATG9AKO cells ) . ( n > 10 , 000 cells ) . ( B ) Representative confocal micrograph ( as maximum intensity projections ) of ATG7KO / TAX1BP1KO K562 cells expressing tf - NBR1 . Selected region ( white box ) of micrograph is shown as single and merged channels from fluorescence microscopy . RFP , magenta ; GFP , green ; merged , white ; Hoechst , blue . Scale bars : large panel , 5 µm ; small panels , 1 µm . ( C ) Correlative light and electron microscopy ( CLEM ) of K562 ATG7KO / TAX1BP1KO cells expressing tf - NBR1 under basal conditions . Analysis workflow is indicated by green arrows . White arrow indicates a structure of interest . White box demarcates zoomed area in bottom images . NBR1 , green ; Hoechst , blue .

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Figure 3(A) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). (n > 10,000 cells).(B) Representative confocal micrograph (as maximum intensity projections) of ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1. Selected region (white box) of micrograph is shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; merged, white; Hoechst, blue. Scale bars: large panel, 5 µm; small panels, 1 µm.(C) Correlative light and electron microscopy (CLEM) of K562 ATG7KO/TAX1BP1KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrow indicates a structure of interest. White box demarcates zoomed area in bottom images. NBR1, green; Hoechst, blue.


Figure 4 ( A ) Wild - type and indicated K562 knock - out cells expressing tf - NBR1 were transfected with TagBFP or TagBFP - RavZ , and BFP - positive cells were analyzed for red : green ratio by flow cytometry . The median red : green ratio of each sample was used to calculate flux relative to WT + TagBFP . Bar graphs represent mean + / - SD from three independent experiments . n > 7 , 500 cells per sample . ( B ) K562 - derived extracts prepared from wild - type ( WT ) and clonal deletion isolates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies . All samples were normalized by total protein using a BCA assay prior to loading . Bar graphs represent mean + / - SD from independent experiments ( n = 4 { NBR1 , NDP52 } ; n = 3 { SQSTM1 } ) . p values for WT vs ATG9AKO was determined using a one sample test ( theoretical mean = 1 ) with Bonferroni correction . ( D ) Wild - type and indicated K562 knock - out cells were treated The corresponding cell extracts were resolved by SDS - PAGE and analyzed by immunoblotting ( IB ) with indicated antibodies . Shown are representative images from one experiment with mean intensity levels from ( E ) indicated below each image ( NBR1 : n = 4 ; TAX1BP1 : n = 3 , NDP52 : n = 4 , tubulin : n = 4 ) . ( E ) Quantitation of protease - protection data from experiments in ( D ) . Bar graphs show the mean + / - SD of each sample from independent experiments . Trypsin - treated samples were compared using a one - way ANOVA ( NBR1 : n = 4 , p = 0 . 0001 ; TAX1BP1 : n = 3 , p = 0 . 0098 ; NDP52 : n = 4 , p < 0 . 0001 ) with Tukey HSD post - test . * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , not

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Figure 4(A) Wild-type and indicated K562 knock-out cells expressing tf-NBR1 were transfected with TagBFP or TagBFP-RavZ, and BFP-positive cells were analyzed for red:green ratio by flow cytometry. The median red:green ratio of each sample was used to calculate flux relative to WT+TagBFP. Bar graphs represent mean +/- SD from three independent experiments. n > 7,500 cells per sample.(B) K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading. Bar graphs represent mean +/- SD from independent experiments (n=4 {NBR1, NDP52}; n=3 {SQSTM1}). p values for WT vs ATG9AKO was determined using a one sample test (theoretical mean = 1) with Bonferroni correction.(D) Wild-type and indicated K562 knock-out cells were treated The corresponding cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with indicated antibodies. Shown are representative images from one experiment with mean intensity levels from (E) indicated below each image (NBR1: n = 4; TAX1BP1: n = 3, NDP52: n = 4, tubulin: n = 4). (E) Quantitation of protease-protection data from experiments in (D). Bar graphs show the mean +/- SD of each sample from independent experiments. Trypsin-treated samples were compared using a one-way ANOVA (NBR1: n = 4, p = 0.0001; TAX1BP1: n = 3, p = 0.0098; NDP52: n = 4, p < 0.0001) with Tukey HSD post-test. **, p < 0.01; *, p < 0.05; ns, not significant


Figure 5 ( A ) Extracts derived from wild - type and deletion K562 cells expressing tf - NBR1 were normalized for total protein by BCA . NBR1 was immunoprecipitated using GFP - Trap dynabeads . Input and eluate were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies . ( B ) Representative confocal micrographs ( as maximum intensity projections ) of wild - type K562 and ATG9AKO cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . In conditions where puncta were not observed , representative cytoplasmic regions were selected to showcase diffuseness of signal . TAX1BP1 , SQSTM1 , or NDP52 , magenta ; NBR1 , green ; Hoechst , blue . ( C ) Shown are representative confocal micrographs ( as maximum intensity projections ) of K562 cells expressing tf - NDP52 ( or tf - TAX1BP1 ) and transduced with sgRNAs targeting ATG9A ( sgATG9A ) or a negative control ( sgControl ) . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence microscopy . RFP , magenta ; GFP , green ; Hoechst , blue .

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Figure 5(A) Extracts derived from wild-type and deletion K562 cells expressing tf-NBR1 were normalized for total protein by BCA. NBR1 was immunoprecipitated using GFP-Trap dynabeads. Input and eluate were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.(B) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 and ATG9AKO cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. In conditions where puncta were not observed, representative cytoplasmic regions were selected to showcase diffuseness of signal. TAX1BP1, SQSTM1, or NDP52, magenta; NBR1, green; Hoechst, blue.(C) Shown are representative confocal micrographs (as maximum intensity projections) of K562 cells expressing tf-NDP52 (or tf-TAX1BP1) and transduced with sgRNAs targeting ATG9A (sgATG9A) or a negative control (sgControl). Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; Hoechst, blue.


Figure 6 ( C ) HEK293T cells were transfected with either myc - TAX1BP1WT or myc - NBR1WT and indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected cells were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input and eluates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies . ( D ) HEK293T cells were transfected with myc - NBR1WT and indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected cells were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input and eluates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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Figure 6(C) HEK293T cells were transfected with either myc-TAX1BP1WT or myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.(D) HEK293T cells were transfected with myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


Figure 7 ( A ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type and deletion K562 cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . P - TBK1 , magenta ; NBR1 , green ; Hoechst , blue . ( B ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type and deletion K562 cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . FIP200 , magenta ; NBR1 , green ; Hoechst , blue . ( C ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO K562 cells expressing tf - NBR1 and transduced with indicated sgRNA . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . FIP200 , magenta ; NBR1 , green ; Hoechst , blue . ( D ) Quantitation of colocalization between NBR1 with FIP200 in wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO cells imaged in ( C ) Bar graphs represent mean + / - SD for three independently generated deletion cell lines ( dots ) . sgATG9A samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - test . * * * , p < 0 . 001 . n > 200 NBR1 puncta for each biological replicate . ( E ) Quantitation of FIP200 intensity at NBR1 puncta in wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO cells imaged in ( C ) . Bar graphs represent mean + / - SD for three independently generated deletion cell lines ( dots ) . n > 80 FIP200 - positive puncta for each biological replicate . Samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - test . * * * , p < 0 . 001 . a . u . , arbitrary intensity units .

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Figure 7(A) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. (B) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue.(C) Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. (D) Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. n > 200 NBR1 puncta for each biological replicate. (E) Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. a.u., arbitrary intensity units.


Figure 1Callus , de novo shoot , and root formation in wild - type ( WT ; Col ) , hag1 - 6 , hag1 - 7 , and 35S : : FLAG : HAG1 hag1 - 7 root explants . Root explants were transferred onto CIM for 1 week ( w ) and then transferred onto fresh CIM , SIM , or RIM for callus , de novo shoot , or root induction , respectively . Scale bar : 1 cm . Graph on the right shows the percentage of explants with shoots formed as scored at 18 days ( d ) on SIM . 36 explants of each genotype were used for scoring . Shoot regeneration assay using Col and hag1 - 6 root explants on media with different cytokinin ( 2IP ) to auxin ( IAA ) ratios . Col and hag1 - 6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg / ml IAA - containing media supplemented with 0 , 500 , or 5 , 000 mg / ml 2IP . Pictures were taken at 27 d after transfer onto each media using representative explants . Scale bar : 2 mm . Root explant - derived calli of Col and hag1 - 6 at 4 w on CIM ( left ) . Scale bar : 1 mm . 4 - w - old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight ( right ) . Histochemical GUS staining of CYCB1 ; 1 : GUS in Col ( left ) and CYCB1 ; 1 : GUS in hag1 - 6 . Shown are root explants at 4 d on CIM . Scale bar : 1 mm . Statistic chart of differentially expressed genes ( DEGs ) in Col and hag1 - 6 roots ( R ) , 4 d CIM - incubated root explants ( C ) , 4 d CIM - incubated + 2 d SIM - incubated root explants ( S ) , and 4 d CIM - incubated + 2 w SIM - incubated root explants ( S2W ) . Green : up - regulated genes , log2 ratio ≥ 1 ; red : down - regulated genes , log2 ratio ≤ - 1 ; FDR < 0 . 01 . Immunoblot analysis of HAG1 : HA protein . Samples are as described in ( E ) . 1 w CIM - incubated Col root explants ( Col ) were used as negative control . Ponceau staining was performed as loading control .

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Figure 1Callus, de novo shoot, and root formation in wild-type (WT; Col), hag1-6, hag1-7, and 35S::FLAG:HAG1 hag1-7 root explants. Root explants were transferred onto CIM for 1 week (w) and then transferred onto fresh CIM, SIM, or RIM for callus, de novo shoot, or root induction, respectively. Scale bar: 1 cm. Graph on the right shows the percentage of explants with shoots formed as scored at 18 days (d) on SIM. 36 explants of each genotype were used for scoring.Shoot regeneration assay using Col and hag1-6 root explants on media with different cytokinin (2IP) to auxin (IAA) ratios. Col and hag1-6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg/ml IAA-containing media supplemented with 0, 500, or 5,000 mg/ml 2IP. Pictures were taken at 27 d after transfer onto each media using representative explants. Scale bar: 2 mm.Root explant-derived calli of Col and hag1-6 at 4 w on CIM (left). Scale bar: 1 mm. 4-w-old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight (right).Histochemical GUS staining of CYCB1;1:GUS in Col (left) and CYCB1;1:GUS in hag1-6. Shown are root explants at 4 d on CIM. Scale bar: 1 mm.Statistic chart of differentially expressed genes (DEGs) in Col and hag1-6 roots (R), 4 d CIM-incubated root explants (C), 4 d CIM-incubated + 2 d SIM-incubated root explants (S), and 4 d CIM-incubated + 2 w SIM-incubated root explants (S2W). Green: up-regulated genes, log2 ratio ≥ 1; red: down-regulated genes, log2 ratio ≤ -1; FDR < 0.01.Immunoblot analysis of HAG1:HA protein. Samples are as described in (E). 1 w CIM-incubated Col root explants (Col) were used as negative control. Ponceau staining was performed as loading control.


Figure 2pWOX5 : : GFP - ER expression in Col ( WT ) and hag1 - 6 explants on CIM and SIM . Time points selected for observation are indicated . Cellular outlines were visualized with propidium iodide ( PI ) staining ( red ) . pSCR : : GFP - ER expression in Col ( WT ) and hag1 - 6 explants on CIM and SIM . Time points selected for observation are indicated . Cellular outlines were visualized with PI staining ( red ) . pSCR : : GFP - ER expression in Col ( WT ) and hag1 - 6 root tips at various time points on CIM . Time points selected for observation are indicated . Cellular outlines were visualized with PI staining ( red ) . WOX5 transcript levels in Col and hag1 - 6 hypocotyl ( D ) and cotyledon ( E ) explants at indicated days on CIM . SCR transcript levels in Col and hag1 - 6 hypocotyl ( F ) and cotyledon ( G ) explants at indicated days on CIM .

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Figure 2pWOX5::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with propidium iodide (PI) staining (red).pSCR::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).pSCR::GFP-ER expression in Col (WT) and hag1-6 root tips at various time points on CIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).WOX5 transcript levels in Col and hag1-6 hypocotyl (D) and cotyledon (E) explants at indicated days on CIM.SCR transcript levels in Col and hag1-6 hypocotyl (F) and cotyledon (G) explants at indicated days on CIM.


Figure 3H3Ac enrichment at the WOX5 ( A ) , SCR ( B ) , PLT1 ( C ) , and PLT2 ( D ) loci in Col ( upper black diagram ) and hag1 - 6 ( lower red diagram ) calli ( 1 w CIM ) as determined by ChIP seq . p - value cutoff for peak detection using MACS2 was 5e - 2 for WOX5 ( A ) and 1e - 5 for SCR ( B ) , PLT1 ( C ) , and PLT2 ( D ) . ChIP - seq data were visualized using Integrated Genome Browser ( IGB ; http : / / bioviz . org / igb / index . html ) . The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position , respectively . Schematic representation of each locus is depicted at the bottom . Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines . 5 ' and 3 ' UTRs are represented as gray boxes . Transcription start sites are indicated with arrows . ChIP - qPCR analysis of H3Ac levels at the WOX5 ( E ) , SCR ( F ) , PLT1 ( G ) , and PLT2 ( H ) loci in Col and hag1 - 6 . Root ( R ) , 1 w CIM ( C ) , and 1 w CIM + 2 w SIM ( S ) samples were used for assays . Direct targeting of HAG1 : HA protein to WOX5 ( I ) , SCR ( J ) , PLT1 ( K ) , and PLT2 ( L ) chromatin as determined by ChIP - qPCR .

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Figure 3H3Ac enrichment at the WOX5 (A), SCR (B), PLT1 (C), and PLT2 (D) loci in Col (upper black diagram) and hag1-6 (lower red diagram) calli (1 w CIM) as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 5e-2 for WOX5 (A) and 1e-5 for SCR (B), PLT1 (C), and PLT2 (D). ChIP-seq data were visualized using Integrated Genome Browser (IGB; http://bioviz.org/igb/index.html). The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position, respectively. Schematic representation of each locus is depicted at the bottom. Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines. 5' and 3' UTRs are represented as gray boxes. Transcription start sites are indicated with arrows.ChIP-qPCR analysis of H3Ac levels at the WOX5 (E), SCR (F), PLT1 (G), and PLT2 (H) loci in Col and hag1-6. Root (R), 1 w CIM (C), and 1 w CIM + 2 w SIM (S) samples were used for assays.Direct targeting of HAG1:HA protein to WOX5 (I), SCR (J), PLT1 (K), and PLT2 (L) chromatin as determined by ChIP-qPCR.


Figure 4Transcript levels of WOX7 ( A ) and WOX14 ( B ) in Col and hag1 - 6 roots ( R ) , 4 d CIM explants ( C ) , 4 d CIM + 2 d SIM explants ( S ) , and 4 d CIM + 2 w SIM explants ( S2W ) . Levels of Col R were set to 1 after normalization with UBQ10 . De novo shoot regeneration of Col , Nossen ( No ) , wox5 - 3 , wox7 - 1 , wox14 - 1 , and wox5 - 3 wox7 - 1 wox14 - 1 root explants . Col is the WT control for wox5 - 3 and wox7 - 1 , whereas No is the WT control for wox14 - 1 . The picture was taken at 3 w on SIM using representative explants of each genotype . Scale bar : 1 cm . Percentage of explants with shoots was scored at indicated weeks on SIM ( D ) . H3Ac enrichment at the WOX14 locus in Col ( upper black diagram ) or hag1 - 6 ( lower red diagram ) calli as determined by ChIP seq . p - value cutoff for peak detection using MACS2 was 1e - 5 . See Figs 3A - D for schematic . ChIP - qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1 - 6 . Regions tested are indicated in the schematic in ( E ) . Sample preparation and data presentation were performed as described in Figs 3E - H . Direct targeting of HAG1 : HA protein to WOX14 chromatin as determined by ChIP - qPCR .

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

Figure 4Transcript levels of WOX7 (A) and WOX14 (B) in Col and hag1-6 roots (R), 4 d CIM explants (C), 4 d CIM + 2 d SIM explants (S), and 4 d CIM + 2 w SIM explants (S2W). Levels of Col R were set to 1 after normalization with UBQ10.De novo shoot regeneration of Col, Nossen (No), wox5-3, wox7-1, wox14-1, and wox5-3 wox7-1 wox14-1 root explants. Col is the WT control for wox5-3 and wox7-1, whereas No is the WT control for wox14-1. The picture was taken at 3 w on SIM using representative explants of each genotype. Scale bar: 1 cm. Percentage of explants with shoots was scored at indicated weeks on SIM (D).H3Ac enrichment at the WOX14 locus in Col (upper black diagram) or hag1-6 (lower red diagram) calli as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 1e-5. See Figs 3A-D for schematic.ChIP-qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1-6. Regions tested are indicated in the schematic in (E). Sample preparation and data presentation were performed as described in Figs 3E-H.Direct targeting of HAG1:HA protein to WOX14 chromatin as determined by ChIP-qPCR.


Figure 5De novo shoot regeneration using Col and scr mutants ; scr - 3 ( A and B ) and scr - 6 ( A and C ) . Explants with shoots were scored at indicated days on SIM using Col and scr - 3 root explants that had been pre - incubated on CIM for 2 w ( B ) or Col and scr - 6 hypocotyl explants that had been pre - incubated on CIM for 1 w ( C ) . Scale bar : 1 mm . De novo shoot regeneration of Col , wox5 - 3 , scr - 3 , and wox5 - 3 scr - 3 . The percentage of root explants with shoots ( E ) and the number of shoots per root explant ( F ) were scored at 15 d on SIM . The explants had been pre - incubated on CIM for 2 w before transfer onto SIM . Scale bar : 1 cm . Transcript levels of PLT1 ( G ) and PLT2 ( H ) in Col , wox5 - 3 , scr - 3 , and wox5 - 3 scr - 3 roots ( R ) , 1 w CIM explants ( C ) , and 1 w CIM + 2 w SIM explants ( S2W ) as determined by RT - qPCR . Levels of Col R were set to 1 after normalization with UBQ10 .

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Figure 5De novo shoot regeneration using Col and scr mutants; scr-3 (A and B) and scr-6 (A and C). Explants with shoots were scored at indicated days on SIM using Col and scr-3 root explants that had been pre-incubated on CIM for 2 w (B) or Col and scr-6 hypocotyl explants that had been pre-incubated on CIM for 1 w (C). Scale bar: 1 mm.De novo shoot regeneration of Col, wox5-3, scr-3, and wox5-3 scr-3. The percentage of root explants with shoots (E) and the number of shoots per root explant (F) were scored at 15 d on SIM. The explants had been pre-incubated on CIM for 2 w before transfer onto SIM. Scale bar: 1 cm.Transcript levels of PLT1 (G) and PLT2 (H) in Col, wox5-3, scr-3, and wox5-3 scr-3 roots (R), 1 w CIM explants (C), and 1 w CIM + 2 w SIM explants (S2W) as determined by RT-qPCR. Levels of Col R were set to 1 after normalization with UBQ10.


Figure 6Activation of WOX5 by DEX treatment of 35S : : GVG - WOX5 in Col and hag1 - 6 . Root explants of 35S : : GVG - WOX5 and 35S : : GVG - WOX5 hag1 - 6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX . DEX was treated for 3 - d - pulse period on CIM or / and SIM ( see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure ) . Morphology of shoots regenerated from 35S : : GVG - WOX5 and 35S : : GVG - WOX5 hag1 - 6 root explants ( A ) . Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX ( B ) . Activation of SCR by DEX treatment of UBI : : mCHERRY - GR - SCR in Col and hag1 - 6 . Activation of WOX5 and SCR by DEX treatment of 35S : : GVG - WOX5 UBI : : mCHERRY - GR - SCR in Col and hag1 - 6 .

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Figure 6Activation of WOX5 by DEX treatment of 35S::GVG-WOX5 in Col and hag1-6. Root explants of 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX. DEX was treated for 3-d-pulse period on CIM or/and SIM (see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure). Morphology of shoots regenerated from 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 root explants (A). Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX (B).Activation of SCR by DEX treatment of UBI::mCHERRY-GR-SCR in Col and hag1-6.Activation of WOX5 and SCR by DEX treatment of 35S::GVG-WOX5 UBI::mCHERRY-GR-SCR in Col and hag1-6.


Figure 1D . Plot of initial MT polymerization velocities at 37 ° C in the presence of increasing concentrations of WT or NN . Tubulin concentration = 5 μM . Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength ( OD500 / min ) . Fitted are sigmoidal dose - response curves : Y = YB + ( ( YT - YB ) / ( 1 + 10Xi - X ) ) , where YB and YT are Y values ( OD500 / min ) at the bottom and top plateaus , respectively , and Xi is the X value at the inflection point . Representative MT samples from 2 : 5 μM WT : tubulin or NN : tubulin incubations are shown in E and F , respectively . MT architecture distributions for WT and NN are shown with pie graphs . E , F . Representative low and high magnification images of MTs nucleated for 10 min at 37 ° C from 5 μM tubulin , in the presence of : 2 μM wild - type DCX ( WT ) ( E ) or 2 μM NDC - NDC ( NN ) ( F ) . H . Representative kymographs with arrowheads indicating MT catastrophes .

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Figure 1D. Plot of initial MT polymerization velocities at 37 °C in the presence of increasing concentrations of WT or NN. Tubulin concentration = 5 μM. Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength (OD500/min). Fitted are sigmoidal dose-response curves: Y = YB + ((YT - YB)/(1 + 10Xi - X)), where YB and YT are Y values (OD500/min) at the bottom and top plateaus, respectively, and Xi is the X value at the inflection point. Representative MT samples from 2:5 μM WT:tubulin or NN:tubulin incubations are shown in E and F, respectively. MT architecture distributions for WT and NN are shown with pie graphs.E, F. Representative low and high magnification images of MTs nucleated for 10 min at 37 °C from 5 μM tubulin, in the presence of: 2 μM wild-type DCX (WT) (E) or 2 μM NDC-NDC (NN) (F).H. Representative kymographs with arrowheads indicating MT catastrophes.


Figure 2A . Representative low - pass filtered cryo - EM micrographs of MTs decorated with wild - type DCX ( WT ) or NDC - NDC construct ( NN ) after 30 sec or 1 h incubation at 37 ° C . Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration . The final ratio of WT or NN to tubulin was 50 : 5 μM , giving dense protein background . WT co - polymerized exclusively 13 - PF MTs , while NN both 13 - and 14 - PF MTs . Rapid ( 30 sec ) MT polymerization in excess of WT produces MTs in an intermediate GTP / GDP . Pi state , whereas long - lived WT - MTs and NN - MTs are all in the GDP state .

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Figure 2A. Representative low-pass filtered cryo-EM micrographs of MTs decorated with wild-type DCX (WT) or NDC-NDC construct (NN) after 30 sec or 1 h incubation at 37 °C. Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration. The final ratio of WT or NN to tubulin was 50:5 μM, giving dense protein background. WT co-polymerized exclusively 13-PF MTs, while NN both 13- and 14-PF MTs. Rapid (30 sec) MT polymerization in excess of WT produces MTs in an intermediate GTP/GDP.Pi state, whereas long-lived WT-MTs and NN-MTs are all in the GDP state.